Construction method and application of novel coronavirus animal infection model

Abstract

The invention discloses a construction method and application of a novel coronavirus animal infection model, and relates to the fields of molecular biology technology and animal gene engineering, the virus replicon prepared by the invention can complete multiple rounds of replication only in specific cells stably expressing novel coronavirus SARS-CoV-2N protein, but cannot be replicated in other cells, and the virus replicon can be used as a novel coronavirus animal infection model. The biological safety risk is greatly reduced, and the system can be operated in a BSL-2 laboratory. Besides, the invention successfully constructs a safe novel coronavirus SARS-CoV-2 animal infection model capable of being used in a BSL-2 laboratory by utilizing an SARS-CoV-2 trans complementary replication subsystem without an N gene and a mouse model for expressing SARS-CoV-2N protein in infected tissue specificity or specific time. The method can be applied to screening, research and development of novel coronavirus resisting drugs.

Description

Technical Field

[0001] The present invention relates to the fields of molecular biology technology and animal genetic engineering, and specifically to a method for constructing and applying a novel coronavirus animal infection model. Background Art

[0002] The novel coronavirus (SARS-CoV-2) is currently raging around the world, causing tens of thousands of infections and deaths worldwide, severely endangering human life. Due to the rapid mutation rate of SARS-CoV-2, a RNA virus, numerous SARS-CoV-2 variants have emerged, such as the novel coronavirus variant Omicron. Compared to the original WH strain, it exhibits significant immune evasion, significantly reducing the effectiveness of currently available drugs and vaccines for SARS-CoV-2. This has created an urgent need for the screening and evaluation of therapeutic drugs, antibodies, and vaccines for SARS-CoV-2. Furthermore, the manipulation of SARS-CoV-2 requires biosafety level 3 (BSL-3) laboratories, significantly increasing the difficulty and cost of drug screening. Several SARS-CoV-2 replicons have been reported that can be operated in biosafety level 2 (BSL-2) laboratories. However, understanding the infection and pathogenesis of SARS-CoV-2 and screening for effective drugs, antibodies, and vaccines requires not only cellular experiments but also extensive in vivo testing in ideal animal models. Mice are one of the most widely used biological models. However, given the numerous physiological and pathological differences between mice and humans, the development of humanized mouse models with functional human genes, cells, or tissues is particularly important. Transgenic animal models, created by genetically modifying the target gene onto mouse chromosomes, are a method for studying some human diseases. Currently, there are no animal models for infection with the novel coronavirus SARS-CoV-2 suitable for use in BSL-2 laboratories.

[0003] Therefore, there is an urgent need to develop a SARS-CoV-2 replicon and its related animal models that can be easily operated in a BSL-2 laboratory.

[0004] In view of this, the present invention is proposed. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for constructing and applying a novel coronavirus animal infection model.

[0006] The present invention is achieved in that:

[0007] In a first aspect, an embodiment of the present invention provides a viral replicon, comprising: a novel coronavirus SARS-Cov-2 genome sequence in which the N gene of the novel coronavirus SARS-Cov-2 is replaced by a reporter gene, wherein the reporter gene comprises a Nanoluc reporter gene and a GFP dual reporter gene.

[0008] In a second aspect, an embodiment of the present invention provides a DNA molecule comprising: a nucleotide sequence capable of rescuing the viral replicon described in the aforementioned embodiment.

[0009] In a third aspect, an embodiment of the present invention provides a viral replicon system, which includes: a cell that stably expresses the novel coronavirus SARS-CoV-2N protein and an expression vector containing the viral replicon described in the preceding embodiment or the DNA molecule described in the preceding embodiment.

[0010] In a fourth aspect, an embodiment of the present invention provides a method for producing a viral replicon, which comprises: culturing cells stably expressing the novel coronavirus SARS-CoV-2 N protein as described in the aforementioned embodiment.

[0011] In a fifth aspect, embodiments of the present invention provide the use of the viral replicon described in the preceding embodiments, or the DNA molecule described in the preceding embodiments, or the viral replicon system described in the preceding embodiments, or the viral replicon generated by the production method described in the preceding embodiments in drug screening against the new coronavirus SARS-Cov-2.

[0012] In a sixth aspect, an embodiment of the present invention provides a DNA fragment comprising: a promoter, loxP-Stop-loxP, SARS-CoV-2N CDS, WPRE and a poly-A gene; the nucleotide sequence of the SARS-CoV-2N CDS is shown in SEQ ID NO: 3.

[0013] In a seventh aspect, an embodiment of the present invention provides a vector comprising the DNA fragment described in the preceding embodiment.

[0014] In an eighth aspect, an embodiment of the present invention provides a method for constructing an animal model, comprising: injecting Cas9 protein or Cas9 mRNA, sgRNA designed for the Tigre site, and the vector described in the preceding embodiment into the cytoplasm or nucleus of a fertilized egg, transplanting it into a surrogate animal mother, and screening the target animals as the F0 generation. The construction method further comprises: mating the F0 generation animals with background animals to obtain the F1 generation.

[0015] In a ninth aspect, embodiments of the present invention provide the use of an animal model obtained by constructing an animal model as described in the preceding embodiments in screening drugs against the novel coronavirus SARS-Cov-2.

[0016] The present invention has the following beneficial effects:

[0017] The viral replicon provided by the present invention completes multiple rounds of replication only in specific cells that stably express the novel coronavirus SARS-CoV-2 N protein, but cannot replicate in other cells. Its biosafety risk is greatly reduced, and it is a system that can be operated in a BSL-2 laboratory. When constructing the replicon, an optimized dual reporter gene is added. The dual reporter gene has strong stability on the genome and high signal expression intensity, which facilitates the observation and detection of subsequent applications of the replicon.

[0018] The replicon system of the present invention can complete replication and package virus-like particles on cells that stably express the N protein, and perform multiple rounds of infection on the cells. This strategy greatly reduces biosafety risks, allowing the system to be operated in a BSL-2 laboratory.

[0019] The animal model construction method provided by this invention features high gene editing efficiency and well-defined integration sites and copy numbers, resolving the issue of variable gene integration sites and copy numbers in conventional random transgenic mice. This model is susceptible to SARS-CoV-2 infection and can be used for research on the development of preventive and therapeutic drugs for COVID-19. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0021] Figure 1 Schematic diagram of the novel coronavirus replicon gene structure and the pCC1-BAC-SARS-CoV-2 vector construction;

[0022] Figure 2 Electrophoresis of SARS-CoV-2 replicon fragments A, B, C, D, E, F, G and CEN / ARS-Trp PCR products;

[0023] Figure 3 Multi-PCR electrophoresis diagram for yeast colony screening and identification;

[0024] Figure 4 It is the mRNA product transcribed from the novel coronavirus replicon genome and the novel coronavirus SARS-CoV-2-N;

[0025] Figure 5This is the GFP signal 16 hours after the novel coronavirus replicon RNA was electroporated;

[0026] Figure 6 This is the GFP signal of Vero-E6-N cells infected with the supernatant of the P0 novel coronavirus replicon for 24 hours;

[0027] Figure 7 Luciferase readouts for the four reporter genes, generations P1-P5;

[0028] Figure 8 To detect the stability of reporter genes in four replicons by RT-PCR;

[0029] Figure 9 The titer of the replicon in different cell lines and cultured with different Trypsin concentrations;

[0030] Figure 10 Schematic diagram of constructing transgenic mice for the novel coronavirus SARS-CoV-2-N;

[0031] Figure 11 The expression levels of hACE2 and SARS-CoV-2N in three SA-CreERT2N-hACE2 mice after induction;

[0032] Figure 12 Schematic diagram of the targeting vector components for animal models;

[0033] Figure 13 This is the PCR electrophoresis result of 3' homology arm and 5' homology arm of F0 generation mice;

[0034] Figure 14 This is the electrophoresis result of tandem detection of F0 generation mice;

[0035] Figure 15 This is the PCR electrophoresis result of the 3' homology arm and 5' homology arm of the background gene hACE2 of F0 generation mice;

[0036] Figure 16 This is the electrophoresis result diagram for identifying F1 generation mice;

[0037] Figure 17 Schematic diagram for constructing mice with inducible N expression using Cre tool mice and SARS-CoV-2-N transgenic mice;

[0038] Figure 18 Sftpc-hACE2-N is a replicon-infected F / F and SA-CreERT2-hACE2-N F / F Body weight changes of mice within seven days;

[0039] Figure 19Sftpc-hACE2-N is a replicon-infected F / F and SA-CreERT2-hACE2-N F / F SARS-CoV-2E RNA levels in the lung and brain tissues of mice on day 7;

[0040] Figure 20 Infect Sftpc-hACE2-N for the replicon F / F and SA-CreERT2-hACE2-N F / F Immunohistochemical images of mouse brain and lung tissues (blue: DAPI, red: hACE2, green: GFP);

[0041] Figure 21 Regenerative therapy for replicon-infected SA-CreERT2-hACE2-N cells with REGEN-COV2 and Paxlovid F / F Body weight changes of mice within seven days;

[0042] Figure 22 Regenerative therapy for replicon-infected SA-CreERT2-hACE2-N cells with REGEN-COV2 and Paxlovid F / F SARS-CoV-2E RNA levels in the lung and brain tissues of mice on day 7;

[0043] Figure 23 Regenerative therapy for replicon-infected SA-CreERT2-hACE2-N cells with REGEN-COV2 and Paxlovid F / F H&E staining of lung and brain tissues of mice on day 7. DETAILED DESCRIPTION

[0044] To make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are used. Where the manufacturer of the reagents or instruments is not specified, all are conventional products that can be purchased commercially.

[0045] On the one hand, an embodiment of the present invention provides a viral replicon, which includes: a novel coronavirus SARS-Cov-2 genome sequence in which the N gene of the novel coronavirus SARS-Cov-2 is replaced by a reporter gene, wherein the reporter gene includes a Nanoluc reporter gene and a GFP dual reporter gene.

[0046] N is a protein necessary for the replication and assembly of coronaviruses. If the N gene is missing, the coronavirus cannot replicate and assemble. As the only non-membrane protein among the structural proteins of coronaviruses, the N protein is easier to express than the other three structural proteins and has less effect on cell morphology. The present invention replaces the N gene on the genome of the new coronavirus SARS-CoV-2 with a dual reporter gene, and at the same time constructs cells that can stably express the N protein. The N protein is trans-complemented in the cell, and the resulting replicon is N gene-deficient. When the cell provides the N protein, it can complete replication and assemble virus-like particles. If the cell does not express the N protein, it cannot replicate and no virus-like particles are produced. This strategy greatly reduces the biosafety risk, allowing this system to be operated in a BSL-2 laboratory.

[0047] During viral replication and passage, the reporter gene is easily lost, resulting in instability of the reporter gene signal, and the longer the reporter gene insert fragment, the more unstable it is. The inventors of this application screened and optimized candidate reporter genes, ensuring the stability of the reporter gene fragment while ensuring signal intensity and sensitivity. The selected reporter gene combination is Enhanced Green Flucrescent Protein (EGFP) and Nanoluc luciferase (Nanoluciferase, Nanoluc), and the two reporter gene sequences are connected by a flexible linker. EGFP is the most widely used fluorescent protein, which is convenient for qualitatively observing and monitoring the replication level of replicons; Nanoluc is a new type of luciferase. Compared with traditional firefly luciferase and sea renilla luciferase, it has a smaller molecular weight (19.1KDa), brighter luminescence and more stable signal, low background, and is suitable for high-throughput detection. It is one of the best performing bioluminescent reporter genes.

[0048] In some embodiments, the replicon further comprises a promoter, which is located upstream of the 5'UTR of the novel coronavirus SARS-Cov-2 genome.

[0049] In some embodiments, the promoter comprises the bacteriophage promoter T7.

[0050] In some embodiments, the nucleotide sequence of the replicon is shown in SEQ ID No: 1:

[0051]

[0052] caacttttaacgtaccaatggaaaaactcaaaacactagttgcaactgcagaagctgaacttgcaaagaatgtgtccttagacaatgtcttatctacttttatttcagcagctcggcaagggtttgttgattcagatgtagaaactaa

[0053] agatgttgttgaatgtcttaaattgtcacatcaatctgacatagaagttactggcgatagttgtaataactatatgctcacctataacaaagttgaaaacatgacaccccgtgaccttggtgcttgtattgactgtagtgcgcgtcatat

[0054] taatgcgcaggtagcaaaaagtcacaacattgctttgatatggaacgttaaagatttcatgtcattgtctgaacaactacgaaaacaaatacgtagtgctgctaaaaagaataacttaccttttaagttgacatgtgcaactactaga

[0055] caagttgttaatgttgtaacaacaaagatagcacttaagggtggtaaaattgttaaataattggttgaagcagttaattaaagttacacttgtgttcctttttgttgctgctattttctatttaataacacctgttcatgtcatgtctaaacatac

[0056] tgacttttcaagtgaaatcataggatacaaggctattgatggtggtgtcactcgtgacatagcatctacagatacttgttttgctaacaaacatgctgattttgacacatggtttagccagcgtggtggtagttatactaatgacaaag

[0057] cttgcccattgattgctgcagtcataacaagagaagtgggttttgtcgtgcctggtttgcctggcacgatattacgcacaactaatggtgactttttgcatttcttacctagagtttttagtgcagttggtaacatctgttacacaccatc

[0058] aaaacttatagagtacactgactttgcaacatcagcttgtgttttggctgctgaatgtacaatttttaaagatgcttctggtaagccagtaccatattgttatgataccaatgtactagaaggttctgttgcttatgaaagtttacgccctg

[0059] acacacgttatgtgctcatggatggctctattattcaatttcctaacacctaccttgaaggttctgttagagtggtaacaacttttgattctgagtactgtaggcacggcacttgtgaaagatcagaagctggtgtttgtgtatctactag

[0060] tggtagatgggtacttaacaatgattattacagatctttaccaggagttttctgtggtgtagatgctgtaaatttatttactaatatgtttacaccactaattcaacctattggtgctttggacatatcagcatctatagtagctggtggtatt

[0061] gtggctatcgtagtaacatgccttgcctactattttatgaggtttagaagagcttttggtgaatacagtcatgtagttgcctttaatactttactattccttatgtcattcattgtactctgtttaacaccagtttactcattcttacctggtgttt

[0062] attctgttatttacttgtacttgacattttatcttactaatgatgtttcttttttagcacatattcagtggatggttatgttcacacctttagtacctttctggataacaattgcttatatcatttgtatttccacaaagcatttctattggttctttagta

[0063] attacctaaagagacgtgtagtctttaatggtgtttcctttagtacttttgaagaagctgcgctgtgcacctttttgttaaataaagaaatgtatctaaagttgcgtagtgatgtgctattacctcttacgcaatataatagatacttagctc

[0064] tttataataagtacaagtattttagtggagcaatggatacaactagctacagagaagctgcttgttgtcatctcgcaaaggctctcaatgacttcagtaactcaggttctgatgttctttaccaaccaccacaaatctctatcacctca

[0065] gctgttttgcagagtggttttagaaaaatggcattcccatctggtaaagttgagggttgtatggtacaagtaacttgtggtacaactacacttaacggtctttggcttgatgacgtagtttactgtccaagacatgtgatctgcacctct

[0066] gaagatatgcttaaccctaattatgaagatttactcattcgtaagtctaatcataatttcttggtacaggctggtaatgttcaactcagggttattggacattctatgcaaaattgtgtacttaagcttaaggttgatacagccaatcctaa

[0067] gacacctaagtataagtttgttcgtattcaaccaggacagactttttcagtgttagcttgttacaatggttcaccatctggtgtttaccaatgtgctatgagacacaatttcactattaagggttcattccttaatggttcatgtggtagtgt

[0068] tggttttaacatagattatgactgtgtctctttttgttacatgcaccatatggaattaccaactggagttcatgctggcacagacttagaaggtaacttttatggaccttttgttgacaggcaaacagcacaagcagctggtacggaca

[0069] caactattacagttaatgttttagcttggttgtacgctgctgttataaatggagacaggtggtttctcaatcgatttaccacaactcttaatgactttaaccttgtggctatgaagtacaattatgaacctctaacacaagaccatgttga

[0070] catactaggacctctttctgctcaaactggaattgccgttttagatatgtgtgcttcattaaaagaattactgcaaaatggtatgaatggacgtaccatattgggtagtgctttattagaagatgaatttacaccttttgatgttgttagac

[0071] aatgctcaggtgttactttccaaagtgcagtgaaaagaacaatcaagggtacacaccactggttgttactcacaattttgacttcacttttagttttagtccagagtactcaatggtctttgttcttttttttgtatgaaaatgcctttttacct

[0072] tttgctatgggtattattgctatgtctgcttttgcaatgatgtttgtcaaacataagcatgcatttctctgtttgtttttgttaccttctcttgccactgtagcttattttaatatggtctatatgcctgctagttgggtgatgcgtattatgacatg

[0073] gttggatatggttgatactagtttgaagctaaaagactgtgttatgtatgcatcagctgtagtgttactaatccttatgacagcaagaactgtgtatgatgatggtgctaggagagtgtggacacttatgaatgtcttgacactcgttta

[0074] taaagtttattatggtaatgctttagatcaagccatttccatgtgggctcttataatctctgttacttctaactactcaggtgtagttacaactgtcatgtttttggccagaggtattgtttttatgtgtgttgagtattgccctattttcttcata

[0075] actggtaatacacttcagtgtataatgctagtttattgtttcttaggctatttttgtacttgttactttggcctcttttgtttactcaaccgctactttagactgactcttggtgtttatgattacttagtttctacacaggagtttagatatatgaat

[0076] tcacagggactactcccacccaagaatagcatagatgccttcaaactcaacattaaattgttgggtgttggtggcaaaccttgtatcaaagtagccactgtacagtctaaaatgtcagatgtaaagtgcacatcagtagtcttactc

[0077] tcagttttgcaacaactcagagtagaatcatcatctaaattgtgggctcaatgtgtccagttacacaatgacattctcttagctaaagatactactgaagcctttgaaaaaatggtttcactactttctgttttgctttccatgcagggtg

[0078] ctgtagacataaacaagctttgtgaagaaatgctggacaacagggcaaccttacaagctatagcctcagagtttagttcccttccatcatatgcagcttttgctactgctcaagaagcttatgaacaggctgttgctaatggtgattc

[0079] tgaagttgttcttaaaaagttgaagaagtctttgaatgtggctaaatctgaatttgaccgtgatgcagccatgcaacgtaagttggaaaagatggctgatcaagctatgacccaaatgtataaacaagctagatctgaggacaaga

[0080] gggcaaaagttactagtgctatgcagacaatgcttttcactatgcttagaaagttggataatgatgcactcaacaacattatcaacaatgcaagagatggttgtgttcccttgaacataatacctcttacaacagcagccaaactaa

[0081] tggttgtcataccagactataacacatataaaaatacgtgtgatggtacaacatttacttatgcatcagcattgtgggaaatccaacaggttgtagatgcagatagtaaaattgttcaacttagtgaaattagtatggacaattcacct

[0082] aatttagcatggcctcttattgtaacagctttaagggccaattctgctgtcaaattacagaataatgagcttagtcctgttgcactacgacagatgtcttgtgctgccggtactacacaaactgcttgcactgatgacaatgcgttagc

[0083] ttactacaacacaacaaagggaggtaggtttgtacttgcactgttatccgatttacaggatttgaaatgggctagattccctaagagtgatggaactggtactatttatacagaactggaaccaccttgtaggtttgttacagacaca

[0084] cctaaaggtcctaaagtgaagtatttatactttattaaaggattaaacaacctaaatagaggtatggtacttggtagtttagctgccacagtacgtctacaagctggtaatgcaacagaagtgcctgccaattcaactgtattatcttt

[0085] ctgtgcttttgctgtagatgctgctaaagcttacaaagattatctagctagtgggggacaaccaatcactaattgtgttaagatgttgtgtacacacactggtactggtcaggcaataacagttacaccggaagccaatatggatca

[0086] agatecctttggtggtgcatcgtgttgtctgtactgccgttgccacatagatcatccaaatcctaaaggattttgtgacttaaaaggtaagtatgtacaaatacctacaacttgtgctaatgaccctgtgggttttacacttaaaaacac

[0087] agtctgtaccgtctgcggtatgtggaaaggttatggctgtagttgtgatcaactccgcgaacccatgcttcagtcagctgatgcacaatcgtttttaaacgggtttgcggtgtaagtgcagcccgtcttacaccgtgcggcacag

[0088] gcactagtactgatgtcgtatacagggcttttgacatctacaatgataaagtagctggttttgctaaattcctaaaaactaattgttgtcgcttccaagaaaaggacgaagatgacaatttaattgattcttactttgtagttaagagac

[0089] acactttctctaactaccaacatgaagaaacaatttataatttacttaaggattgtccagctgttgctaaacatgacttctttaagtttagaatagacggtgacatggtaccacatatatcacgtcaacgtcttactaaatacacaatgg

[0090] cagacctcgtctatgctttaaggcattttgatgaaggtaattgtgacacattaaaagaaatacttgtcacatacaattgttgtgatgatgattatttcaataaaaaggactggtatgattttgtagaaaacccagatatattacgcgtata

[0091] cgccaacttaggtgaacgtgtacgccaagctttgttaaaaacagtacaattctgtgatgccatgcgaaatgctggtattgttggtgtactgacattagataatcaagatctcaatggtaactggtatgatttcggtgatttcatacaaa

[0092] ccacgccaggtagtggagttcctgttgtagattcttattattcattgttaatgcctatattaaccttgaccagggctttaactgcagagtcacatgttgacactgacttaacaaagccttacattaagtgggatttgttaaaatatgacttc

[0093] acggaagagaggttaaaactctttgaccgttattttaaatattgggatcagacataccacccaaattgtgttaactgtttggatgacagatgcattctgcattgtgcaaactttaatgttttattctctacagtgttcccacttacaagtttt

[0094] ggaccactagtgagaaaaatatttgttgatggtgttccatttgtagtttcaactggataccacttcagagagctaggtgttgtacataatcaggatgtaaacttacatAGCTCtagacttagtttTAAGGAATtacttgtgta

[0095] tgctgctgaccctgctatgcacgctgcttctggtaatctattactagataaacgcactacgtgcttttcagtagctgcacttactaacaatgttgcttttcaaactgtcaaacccggtaattttaacaaagacttctatgactttgctgtgtgt

[0096] ctaagggtttctttaaggaaggaagttctgttgaattaaaacacttcttctttgctcaggatggtaatgctgctatcagcgattatgactactatcgttataatctaccaacaatgtgtgatatcagacaactactatttgtagttgaagtt

[0097] gttgataagtactttgattgttacgatggtggctgtattaatgctaaccaagtcatcgtcaacaacctagacaaatcagctggttttccatttaataaatggggtaaggctagactttattatgattcaatgagttgtgaggatcaagat

[0098] gcacttttcgcatatacaaaacgtaatgtcatccctactataactcaaatgaatcttaagtatgccattagtgcaaagaatagagctcgcaccgtagctggtgtctctatctgtagtactatgaccaatagacagtttcatcaaaaatt

[0099] attgaaatcaatagccgccactagaggagctactgtagtaattggaacaagcaaattctatggtggttggcacaacatgttaaaaactgtttatagtgatgtagaaaaccctcaccttatgggttgggattatcctaaatgtgatag

[0100] agccatgcctaacatgcttagaattatggcctcacttgttcttgctcgcaaacatacaacgtgttgtagcttgtcacaccgtttctatagattagctaatgagtgtgctcaagtattgagtgaaatggtcatgtgtggcggttcactata

[0101] tgttaaaccaggtggaacctcatcaggagatgccacaactgcttatgctaatagtgtttttaacatttgtcaagctgtcacggccaatgttaatgcacttttatctactgatggtaacaaaattgccgataagtatgtccgcaatttaca

[0102] acacagactttatgagtgtctctatagaaatagagatgttgacacagactttgtgaatgagttttacgcatatttgcgtaaacatttctcaatgatgatactttctgacgatgctgttgtgtgtttcaatagcacttatgcatctcaaggtct

[0103] agtggctagcataaagaactttaagtcagttctttattatcaaaacaatgtttttatgtctgaagcaaaatgttggactgagactgaccttactaaaggacctcatgaattttgctctcaacatacaatgctagttaaacagggtgatga

[0104] ttatgtgtaccttccttacccagatccatcaagaatcctaggggccggctgttttgtagatgatatcgtaaaaacagatggtacacttatgattgaacggttcgtgtctttagctatagatgcttacccacttactaaacatcctaatca

[0105] ggagtatgctgatgtctttcatttgtacttacaatacataagaaagctacatgatgagttaacaggacacatgttagacatgtattctgttatgcttactaatgataacacttcaaggtattgggaacctgagttttatgaggctatgtac

[0106] acaccgcatacagtcttacaggctgttggggcttgtgttctttgcaattcacagacttcattaagatgtggtgcttgcatacgtagaccattcttatgttgtaaatgctgttacgaccatgtcatatcaacatcacataaattagtcttgtc

[0107] tgttaatccgtatgtttgcaatgctccaggttgtgatgtcacagatgtgactcaactttacttaggaggtatgagctattattgtaaatcacataaaccacccattagttttccattgtgtgctaatggacaagtttttggtttatataaaaa

[0108] tacatgtgttggtagcgataatgttactgactttaaatgcaattgcaacatgtgactggacaaatgctggtgattacattttagctaacacctgtactgaaagactcaagctttttgcagcagaaacgctcaaagctactgaggagac

[0109] atttaaactgtcttatggtattgctactgtacgtgaagtgctgtctgacagagaattacatctttcatgggaagttggtaaacctagaccaccacttaaccgaaattatgtctttactggttatcgtgtaactaaaaacagtaaagtaca

[0110] aataggagagtacacctttgaaaaaggtgactatggtgatgctgttgtttaccgaggtacaacaacttacaaattaaatgttggtgattattttgtgctgacatcacatacagtaatgccattaagtgcacctacactagtgccacaa

[0111] gagcactatgttagaattactggcttatacccaacactcaatatctcagatgagttttctagcaatgttgcaaattatcaaaaggttggtatgcaaaagtattctacactccagggaccacctggtactggtaagagtcattttgctatt

[0112] ggcctagctctctactacccttctgctcgcatagtgtatacagcttgctctcatgccgctgttgatgcactatgtgagaaggcattaaaatatttgcctatagataaatgtagtagaattatacctgcacgtgctcgtgtagagtgtttt

[0113] gataaattcaaagtgaattcaacattagaacagtatgtcttttgtactgtaaatgcattgcctgagacgacagcagatatagttgtctttgatgaaatttcaatggccacaaattatgatttgagtgttgtcaatgccagattatgtgcta

[0114] agcactatgtgtacattggcgaccctgctcaattacctgcaccacgcacattgctaactaagggcacactagaaccagaatatttcaattcagtgtgtagacttatgaaaactataggtccagacatgttcctcggaacttgtcgg

[0115] cgttgtcctgctgaaattgttgacactgtgagtgctttggtttatgataataagcttaaagcacataaagacaaatcagctcaatgctttaaaatgttttataagggtgttatcacgcatgatgtttcatctgcaattaacaggccacaa

[0116] ataggcgtggtaagagaattccttacacgtaaccctgcttggagaaaagctgtctttatttcaccttataattcacagaatgctgtagcctcaaagattttgggactaccaactcaaactgttgattcatcacagggctcagaatatg

[0117] actatgtcatattcactcaaaccactgaaacagctcactcttgtaatgtaaacagatttaatgttgctattaccagagcaaaagtaggcatactttgcataatgtctgatagagacctttatgacaagttgcaatttacaagtcttgaaa

[0118] ttccacgtaggaatgtggcaactttacaagctgaaaatgtaacaggactctttaaagattgtagtaaggtaatcactgggttacatcctacacaggcacctacacacctcagtgttgacactaaattcaaaactgaaggtttatgtg

[0119] ttgacgtacctggcatacctaaggacatgacctatagaagactcatctctatgatgggttttaaaatgaattatcaagttaaatggttaccctaacatgtttatcacccgcgaagaagctataagacatgtacgtgcatggattggctt

[0120] cgatgtcgaggggtgtcatgctactagagaagctgttggtaccaatttacctttacagctaggtttttctacaggtgttaacctagttgctgtacctacaggttatgttgatacacctaataatacagatttttccagagttagtgctaaa

[0121] ccaccgcctggagatcaatttaaacacctcataccacttatgtacaaaggacttccttggaatgtagtgcgtataaagattgtacaaatgttaagtgacacacttaaaatctctctgacagagtcgtatttgtcttatgggcacatg

[0122] gctttgagttgacatctatgaagtattttgtgaaaataggacctgagcgcacctgttgtctatgtgatagacgtgccacatgcttttccactgcttcagacacttatgcctgttggcatcattctattggatttgattacgtctataatccg

[0123] tttatgattgatgttcaacaatggggttttacaggtaacctacaaagcaaccatgatctgtattgtcaagtccatggtaatgcacatgtagctagttgtgatgcaatcatgactaggtgtctagctgtccacgagtgctttgttaagcgt

[0124] gttgactggactattgaatatcctataattggtgatgaactgaagattaaatgcggcttgtagaaaggttcaacacatggttgttaaagctgcattattagcagacaaattcccagttcttcacgacattggtaaccctaaagctattaa

[0125] gtgtgtacctcaagctgatgtagaatggaagttctatgatgcacagccttgtagtgacaaagcttataaaaatagaaagaatttctattcttatgccacacattctgacaaattcacagatggtgtatgcctattttggaattgcaatgt

[0126] cgatagatatcctgctaattccattgtttgtagatttgacactagagtgctatctaaccttaacttgcctggttgtgatggtggcagtttgtatgtaaataaacatgcattccacacaccagcttttgataaaagtgcttttgttaatttaaa

[0127] acaattaccatttttctattactctgacagtccatgtgagtctcatggaaaacaagtagtgtcagatatagattatgtaccactaaagtctgctacgtgtataacacgttgcaatttaggtggtgctgtctgtagacatcatgctaatga

[0128] gtacagattgtatctcgatgcttataacatgatgatctcagctggctttagcttgtgggtttacaaacaatttgatacttataacctctggaacacttttacaaagacttcagagtttagaaaatgtggcttttaatgttgtaaataagggac

[0129] actttgatggacaacagggtgaagtaccagtttctatcattaataacactgtttacacaaaagttgatggtgttgatgtagaattgtttgaaaataaaacaacattacctgttaatgtagcatttgagctttgggctaagcgcaacatta

[0130] aaccagtaccagaggtgaaaatactcaataatttgggtgtggacattgctgctaatactgtgatctgggactacaaaagagatgctccagcacatatatctactattggtgtttgttctatgactgacatagccaagaaaccaattg

[0131] aaacgatttgtgcaccactcactgtcttttttgatggtagagttgatggtcaagtagacttatttagaaatgcccgtaatggtgttcttattacagagggtagtgttaaaggtttacaaccatctgtaggtcccaaacaagctagtctta

[0132] atggagtcacattaattggagaagccgtaaaaacacagttcaattattataagaaagttgatggtgttgtccaacaattacctgaaacttactttactcagagtagaaatttacaagaatttaaacccaggagtcaaatggaaattga

[0133] tttcttagaattagctatggatgaattcattgaacggtataaattagaaggctatgccttcgaacatatcgtttatggagattttagtcatagtcagttaggtggtttacatctactgattggactagctaaacgttttaaggaatcaccttt

[0134] tgaattagaagattttattcctatggacagtacagttaaaaactatttcataacagatgcgcaaacaggttcatctaagtgtgtgtgttctgttattgatttattacttgatgattttgttgaaataataaaatcccaagatttatctgtagttt

[0135] ctaaggttgtcaaagtgactattgactatacagaaatttcatttatgctttggtgtaaagatggccatgtagaaacattttacccaaattacaatctagtcaagcgtggcaaccgggtgttgctatgcctaatctttacaaaatgcaa

[0136] agaatgctattagaaaagtgtgaccttcaaaattatggtgatagtgcaacattacctaaaggcataatgatgaatgtcgcaaatatactcaactgtgtcaatatttaaacacattaacattagctgtaccctataatatgagagttata

[0137] cattttggtgctggttctgataaaggagttgcaccaggtacagctgttttaagacagtggttgcctacgggtacgctgcttgtcgattcagatcttaatgactttgtctctctgatgcagattcaactttgattggtgattgtgcaactgtac

[0138] atacagctaataaatgggatctcattattagtgatatgtacgaccctaagactaaaaatgttacaaaagaaaatgactctaaagagggttttttcacttacatttgtgggtttatacaacaaaagctagctcttggaggttccgtggct

[0139] ataaagataacagaacattcttggaatgctgatctttataagctcatgggacacttcgcatggtggacagcctttgttactaatgtgaatgcgtcatcatctgaagcatttttaattggatgtaattatcttggcaaaccacgcgaaca

[0140] aatagatggttatgtcatgcatgcaaattacatattttggaggaatacaaatccaattcagttgtcttcctattctttatttgacatgagtaaatttccccttaaattaaggggtactgctgttatgtctttaaaagaaggtcaaatcaatga

[0141] tatgattttatctcttcttagtaaaggtagacttataattagagaaaacaacagagttgttatttctagtgatgttcttgttaacaactaaacgaacaatgtttgtttttcttgttttattgccactagtctctagtcagtgtgttaatcttataac

[0142] cagaactcaatcatacactaattctttcacacgtggtgtttattaccctgacaaagttttcagatcctcagttttacattcaactcaggacttgttcttacctttcttttccaatgttacttggttccatgctatctctgggaccaatggtacta

[0143] agaggtttgataaccctgtcctaccatttaatgatggtgtttattttgcttccactgagaagtctaacataataagaggctggatttttggtactactttagattcgaagacccagtccctacttattgttaataacgctactaatgttgttat

[0144] taaagtctgtgaatttcaattttgtaatgatccatttttggatgtttattaccacaaaaacaacaaaagttggatggaaagtgagttcagagtttattctagtgcgaataattgcacttttgaatatgtctctcagccttttcttatggacctt

[0145] gaaggaaaacagggtaatttcaaaaatcttagggaatttgtgtttaagaatattgatggttattttaaaatatattctaagcacacgcctattaatttagggcgtgatctccctcagggtttttcggctttagaaccattggtagatttgcc

[0146] aataggtattaacatcactaggtttcaaactttacttgctttacatagaagttatttgactcctggtgattcttcttcaggttggacagctggtgctgcagcttattatgtgggttatcttcaacctaggacttttctattaaaatataatgaa

[0147] aatggaaccattacagatgctgtagactgtgcacttgaccctctctcagaaacaaagtgtacgttgaaatccttcactgtagaaaaaggaatctatcaaacttctaactttagagtccaaccaacagaatctattgttagatttcctaa

[0148] tattacaaacttgtgcccttttgatgaagtttttaacgccaccagatttgcatctgtttatgcttggaacaggaagagaatcagcaactgtgttgctgattattctgtcctatataatttcgcaccatttttcgcttttaagtgttatggagtg

[0149] tctcctactaaattaaatgatctctgctttactaatgtctatgcagattcatttgtaattagaggtaatgaagtcagccaaatcgctccagggcaaactggaaatattgctgattataattataaattaccagatgattttacaggctgcgt

[0150] tatagcttggaattctaacaagcttgattctaaggttggtggtaattataattaccggtatagattgtttaggaagtctaatctcaaaccttttgagagagatatttcaactgaaatctatcaggccggtaacaaaccttgtaatggtgtt

[0151] gcaggtgttaattgttactttcctttacaatcatatggtttccgacccacttatggtgttggtcaccaaccatacagagtagtagtactttcttttgaacttctacatgcaccagcaactgtttgtggacctaaaaagtctactaatttggtt

[0152] aaaaacaaatgtgtcaatttcaacttcaatggtttaacaggcacaggtgttcttactgagtctaacaaaaagtttctgcctttccaacaatttggcagagacattgctgacactactgatgctgtccgtgatccacagacacttgaga

[0153] ttcttgacattacaccatgttcttttggtggtgtcagtgttataacaccaggaacaaatacttctaaccaggttgctgttctttatcagggtgttaactgcacagaagtccctgttgctattcatgcagatcaacttactcctacttggcgt

[0154] gtttattctacaggttctaatgtttttcaaacacgtgcaggctgtttaataggggctgaatatgtcaacaactcatatgagtgtgacatacccattggtgcaggtatatgcgctagttatcagactcagactaagtctcatcggcgggc

[0155] acgtagtgtagctagtcaatccatcattgcctacactatgtcacttggtgcagaaaattcagttgcttactctaataactctattgccatacccacaaattttactattagtgttaccacagaaattctaccagtgtctatgaccaagaca

[0156] tcagtagattgtacaatgtacatttgtggtgattcaactgaatgcagcaatcttttgttgcaatatggcagtttttgtacacaattaaaacgtgctttaactggaatagctgttgaacaagacaaaaacacccaagaagtttttgcacaa

[0157] gtcaaacaaatttacaaaacaccaccaattaaatattttggtggttttaatttttcacaaatattaccagatccatcaaaaccaagcaagaggtcatttattgaagatctacttttcaacaaagtgacacttgcagatgctggcttcatca

[0158] aacaatatggtgattgccttggtgatattgctgctagagacctcatttgtgcacaaaagtttaacggccttactgttttgccacctttgctcacagatgaaatgattgctcaatacacttctgcactgttagcgggtacaatcacttctg

[0159] gttggacctttggtgcaggtgctgcattacaaataccatttgctatgcaaatggcttataggtttaatggtattggagttacacagaatgttctctatgagaaccaaaaattgattgccaaccaatttaatagtgctattggcaaaattc

[0160] aagactcactttcttccacagcaagtgcacttggaaaacttcaagatgtggtcaaccataatgcacaagctttaaacacgcttgttaaacaacttagctccaaatttggtgcaatttcaagtgttttaaatgatatcctttcacgtcttg

[0161] acaaagttgaggctgaagtgcaaattgataggttgatcacaggcagacttcaaagtttgcagacatatgtgactcaacaattaattagagctgcagaaatcagagcttctgctaatcttgctgctactaaaatgtcagagtgtgtac

[0162] ttggacaatcaaaaagagttgattttgtggaaagggctatcatcttatgtccttccctcagtcagcacctcatggtgtagtcttcttgcatgtgacttatgtccctgcacaagaaaagaacttcacaactgctcctgccatttgtcatg

[0163] atggaaaagcacactttcctcgtgaaggtgtctttgtttcaaatggcacacactggtttgtaacacaaaggaatttttatgaaccacaaatcattactacagacaacacatttgtgtctggtaactgtgatgttgtaataggaattgtca

[0164] acaacacagtttatgatcctttgcaacctgaattagattcattcaaggaggagttagataaatattttaagaatcatacatcaccagatgttgatttaggtgacatctctggcattaatgcttcagttgtaaacattcaaaaagaaattga

[0165] ccgcctcaatgaggttgccaagaatttaaatgaatctctcatcgatctccaagaacttggaaagtatgagcagtatataaaatggccatggtacatttggctaggttttatagctggcttgattgccatagtaatggtgacaattatg

[0166] ctttgctgtatgaccagttgctgtagttgtctcaagggctgttgttcttgtggatcctgctgcaaatttgatgaagacgactctgagccagtgctcaaaggagtcaaattacattacacataaacgaacttatggatttgtttatgagaa

[0167] tcttcacaattggaactgtaactttgaagcaaggtgaaatcaaggatgctactccttcagattttgttcgcgctactgcaacgataccgatacaagcctcactccctttcggatggcttattgttggcgttgcacttcttgctgtttttca

[0168] gagcgcttccaaaatcataactctcaaaaagagatggcaactagcactctccaagggtgttcactttgtttgcaacttgctgttgttgtttgtaacagtttactcacaccttttgctcgttgctgctggccttgaagccccttttctctatc

[0169] tttatgctttagtctacttcttgcagagtataaactttgtaagaataatgaggctttggctttgctggaaatgccgttccaaaaacccattactttatgatgccaactattttctttgctggcatactaattgttacgactattgtatacct

[0170] tacaatagtgtaacttcttcaattgtcattacttcaggtgatggcacaacaagtcctatttctgaacatgactaccagattggtggttatactgaaaaatgggaatctggagtaaaagactgtgttgtattacacagttacttcacttca

[0171] gactattaccagctgtactcaactcaattgagtacagacattggtgttgaacatgttaccttcttcatctacaataaaattgttgatgagcctgaagaacatgtccaaattcacacaatcgacggttcatccggagttgttaatccagt

[0172] aatggaaccaaatttatgatgaaccgacgacgactactagcgtgcctttgtaagcacaagctgatgagtacgaacttatgtactcattcgtttcggaagagataggtacgttaatagttaatagcgtacttctttttcttgctttcgtggt

[0173] attcttgctagttacactagccatccttactgcgcttcgattgtgtgcgtactgctgcaatattgttaacgtgagtcttgtaaaaccttctttttacgtttactctcgtgttaaaaatctgaattcttctagagttcctgatcttctggtctaaac

[0174] gaactaaatattatattagtttttctgtttggaactttaattttagccatggcaaattccaacggtactattaccgttgaagagcttaaaaagctccttgaagaatggaacctagtaataggtttcctattccttacatggatttgtcttctac

[0175] aatttgcctatgccaacaggaataggtttttgtatataattaagttaattttcctctggctgttatggccagtaactttaacttgttttgtgcttgctgctgtttacagaataaattggatcaccggtggaattgctatcgcaatggcttgtctt

[0176] gtaggcttgatgtggctcagctacttcattgcttctttcagactgtttgcgcgtacgcgttccatgtggtcattcaatccagaaactaacattcttctcaacgtgccactccatggcactattctgaccagaccgcttctagaaagtga

[0177] actcgtaatcggagctgtgatccttcgtggacatcttcgtattgctggacaccatctaggacgctgtgacatcaaggacctgcctaaagaaatcactgttgctacatcacgaacgctttcttattacaaattgggagcttcgcagcg

[0178] tgtagcaggtgactcaggttttgctgcatacagtcgctacaggattggcaactataaattaaacacagaccattccagtagcagtgacaatattgctttgcttgtacagtaagtgacaacagatgtttcatctcgttgactttcaggtt

[0179] actatagcagagatattactaattattatgaggacttttaaagtttccatttggaatcttgattacatcataaacctcataattaaaaatttatctaagtcactaactgagaataaatattctcaattagatgaagagcaaccaatggagat

[0180] tgattaaacgaacatgaaaattattcttttcttggcactgataacactcgctacttgtgagctttatcactaccaagagtgtgttagaggtacaacagtacttttaaaagaaccttgctcttctggaacatacgagggcaattcaccatt

[0181] tcatcctctagctgataacaaatttgcactgacttgctttagcactcaatttgcttttgcttgtcctgacggcgtaaaacacgtctatcagttacgtgccagatcagtttcacctaaactgttcatcagacaagaggaagttcaagaact

[0182] ttactctccaatttttcttattgttgcggcaatagtgtttataacactttgcttcacactcaaaagaaagacagaatgattgaactttcattaattgacttctatttgtgctttttagcctttctgttattccttgttttaattatgcttattatcttttg

[0183] gttctcacttgaactgcaagatcataatgaaacttgtcacgcctaaatgaacatgaaatttcttgttttcttaggaatcatcacaactgtagctgcatttcaccaagaatgtagtttacagtcatgtactcaacatcaaccatatgtagtt

[0184] gatgacccgtgtcctattcacttctattctaaatggtatattagagtaggagctagaaaaatcagcacctttaattgaattgtgcgtggatgaggctggttctaaatcacccattcagtacatcgatatcggtaattatacagtttcctgtt

[0185] taccttttacaattaattgccaggaacctaaattgggtagtcttgtagtgcgttgttcgttctatgaagactttttagagtatcatgacgttcgtgttgttttagatttcatctaaacgaacaaacttaaatggtcttcacactcgaagatttc

[0186] gttggggactggcgacagacagccggctacaacctggaccaagtccttgaacagggaggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaatgggctgaaga

[0187] tcgacatccatgtcatcatcccgtatgaaggtctgagcggcgaccaaatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtgatcctgcactatggcacactggtaatcgacggggt

[0188] tacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaacagggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccct

[0189] gctgttccgagtaaccatcaacggagtgaccggctggcggctgtgcgaacgcattctggcgggctctagcggcatgggcgtgatcaagcccgacatgaagatcaagctgcggatggagggcgccgtgaacggccacaa

[0190] attcgtgatcgagggcgacgggaaaggcaagccctttgagggtaagcagactatggacctgaccgtgatcgagggcgcccccctgcccttcgcttatgacattctcaccaccgtgttcgactacggtaaccgtgtcttcgcc

[0191] aagtaccccaaggacatccctgactacttcaagcagaccttccccgagggctactcgtgggagcgaagcatgacatacgaggaccagggaatctgtatcgctacaaacgacatcaccatgatgaagggtgtggacgactg

[0192] cttcgtgtacaaaatccgcttcgacggggtcaacttccctgctaatggcccggtgatgcagcgcaagaccctaaagtgggagcccagtaccgagaagatgtacgtgcgggacggcgtactgaagggcgatgttaatatggc

[0193] actgctcttggagggaggcggccactaccgctgcgacttcaagaccacctacaaagccaagaaggtggtgcagcttcccgactaccacttcgtggaccaccgcatcgagatcgtgagccacgacaaggactacaacaaa

[0194] gtcaagctgtacgagcacgccgaagcccacagcggactaccccgccaggccggctaaactcatgcagaccacaaggcagatgggctatataaacgttttcgcttttccgtttacgatatatagtcta ctcttgtgcagaatgaattctcgtaactacatagcacaagtagatgtagttaactttaatctcacatagcaatctttaatcagtgtgtaacattagggaggacttgaaagagccaccacattttcacc.

[0195] The sequence shown in SEQ ID NO: 1 is the sequence of the replicon, in which T is the same as U. Because it is a DNA sequence constructed into the plasmid, U is replaced by T when the sequence is provided.

[0196] On the other hand, an embodiment of the present invention provides a DNA molecule comprising: a nucleotide sequence capable of successfully rescuing the viral replicon described in any of the aforementioned embodiments.

[0197] The "virus rescue" in this article is the same as (the rescue of virus), which is a reverse genetic manipulation technology, which refers to the use of certain methods to enable the virus in a non-proliferative infection to complete the replication and proliferation process and produce progeny viruses.

[0198] In some embodiments, the DNA molecule is a vector containing a replicon nucleotide sequence, which can express and clone the above-mentioned viral replicon.

[0199] In some embodiments, the DNA molecule comprises the nucleotide sequence shown in SEQ ID NO: 2:

[0200]

[0201] cgaattttgtggcactgagaatttgactaaagaaggtgccactacttgtggttacttaccccaaaatgctgttgttaaaatttattgtccagcatgtcacaattcagaagtaggacctgagcatagtcttgccgaataccataatgaat

[0202] ctggcttgaaaaccattcttcgtaagggtggtcgcactattgcctttggaggctgtgtgttctcttatgttggttgccataacaagtgtgcctattgggttccacgtgctagcgctaacataggttgtaaccatacaggtgttgttgga

[0203] gaaggttccgaaggtcttaatgacaaccttcttgaaatacttcaaaaagagaaagtcaacatcaatattgttggtgactttaaacttaatgaagagatcgccattattttggcatctttttctgcttccacaagtgcttttgtggaaactg

[0204] tgaaaggtttggattataaagcattcaaacaaattgttgaatcctgtggtaattttaaagttacaaaaggaaaagctaaaaaaggtgcctggaatattggtgaacagaaatcaatactgagtcctctttatgcatttgcatcagaggc

[0205] tgctcgtgttgtacgatcaattttctcccgcactcttgaaactgctcaaaattctgtgcgtgttttacagaaggccgctataacaatactagatggaatttcacagtattcactgagactcattgatgctatgatgttcacatctgatttgg

[0206] ctactaacaatctagttgtaatggcctacattacaggtggtgttgttcagttgacttcgcagtggctaactaacatctttggcactgtttatgaaaaactcaaacccgtccttgattggcttgaagagaagtttaaggaaggtgtagag

[0207] tttcttagagacggttgggaaattgttaaatttatctcaacctgtgcttgtgaaattgtcggtggacaaattgtcacctgtgcaaaggaaattaaggagagtgttcagacattctttaagcttgtaaataaatttttggctttgtgtgctga

[0208] ctctatcattattggtggagctaaacttaaagccttgaatttaggtgaaacatttgtcacgcactcaaagggattgtacagaaagtgtgttaaatccagagaagaaactggcctactcatgcctctaaaagccccaaaagaaattat

[0209] cttcttagagggagaaacacttcccacagaagtgttaacagaggaagttgtcttgaaaactggtgatttacaaccattagaacaacctactagtgaagctgttgaagctccattggttggtacaccagtttgtattaacgggcttat

[0210] gttgctcgaaatcaaagacacagaaaagtactgtgcccttgcacctaatatgatggtaacaaacaataccttcacactcaaaggcggtgcaccaacaaaggttacttttggtgatgacactgtgatagaagtgcaaggttacaa

[0211] gagtgtgaatatcatttttgaacttgatgaaaggattgataaagtacttaatgagaagtgctctgcctatacagttgaactcggtacagaagtaaatgagttcgcctgtgttgtggcagatgctgtcataaaaactttgcaaccagtat

[0212] ctgaattacttacaccactgggcattgatttagatgagtggagtatggctacatactacttatttgatgagtctggtgagtttaaattggcttcacatatgtattgttctttttaccctccagatgaggatgaagaagaaggtgattgtga

[0213] agaagaagagtttgagccatcaactcaatatgagtatggtactgaagatgattaccaaggtaaacctttggaatttggtgccacttctgctgctcttcaacctgaagaagagcaagaagaagattggttagatgatgatagtcaac

[0214] aaactgttggtcaacaagacggcagtgaggacaatcagacaactactattcaaacaattgttgaggttcaacctcaattagagatggaacttacaccagttgttcagactattgaagtgaatagttttagtggttatttaaaacttact

[0215] gacaatgtatacattaaaaatgcagacattgtggaagaagctaaaaaggtaaaaccaacagtggttgttaatgcagccaatgtttaccttaaacatggaggaggtgttgcaggagccttaaataaggctactaacaatgccatg

[0216] caagttgaatctgatgattacatagctactaatggaccacttaaagtgggtggtagttgtgttttaagcggacacaatcttgctaaacactgtcttcatgttgtcggcccaaatgttaacaaaggtgaagacattcaacttcttaagag

[0217] tgcttatgaaaattttaatcagcacgaagttctacttgcaccattattatcagctggtatttttggtgctgaccctatacattctttaagagtttgtgtagatactgttcgcacaaatgtctacttagctgtctttgataaaaatctctatgaca

[0218] aacttgtttcaagctttttggaaatgaagagtgaaaagcaagttgaacaaaagatcgctgagattcctaaagaggaagttaagccatttataactgaaagtaaaccttcagttgaacagagaaaacaagatgataagaaaatcaa

[0219] agcttgtgttgaagaagttacaacaactctggaagaaactaagttcctcacagaaaacttgttactttatattgacattaatggcaatcttcatccagattctgccactcttgttagtgacattgacatcactttcttaaagaaagatgct

[0220] ccatatatagtgggtgatgttgttcaagagggtgttttaactgctgtggttatacctactaaaaaggctagtggcactactgaaatgctagcgaaagctttgagaaaagtgccaacagacaattatataaccacttacccgggtca

[0221] gggtttaaatggttacactgtagaggaggcaaagacagtgcttaaaaagtgtaaaagtgctttttacattctaccatctattatctctaatgagaagcaagaaattcttggaactgtttcttggaatttgcgagaaatgcttgcacatg

[0222] cagaagaaacacgcaaattaatgcctgtctgtgtggaaactaaagccatagtttcaactatacagcgtaaatataagggtattaaaatacaagagggtgtggttgattatggtgctagattttacttttacaccagtaaaacaactgt

[0223] agcgtcacttatcaacacacttaacgatctaaatgaaactcttgttacaatgccacttggctatgtaacacatggcttaaatttggaagaagctgctcggtatatgagatctctcaaagtgccagctacagtttctgtttcttcacctga

[0224] tgctgttacagcgtataatggttatcttacttcttcttctaaaacacctgaagaacattttattgaaaccatctcacttgctggttcctataaagattggtcctattctggacaatctacacaactaggtatagaatttcttaagagaggtg

[0225] ataaaagtgtatattacactagtaatcctaccacattccacctagatggtgaagttatcacctttgacaatcttaagacacttctttctttgagagaagtgaggactattaaggtgtttacaacagtagacaacattaacctccacacg

[0226] caagttgtggacatgtcaatgacatatggacaacagtttggtccaacttatttggatggagctgatgttactaaaataaaacctcataattcacatgaaggtaaaacattttatgttttacctaatgatgacactctacgtgttgaggct

[0227] tttgagtactaccacacaactgatcctagttttctgggtaggtacatgtcagcattaaatcacactaaaaagtggaaatacccacaagttaaatggtttaacttctattaaatgggcagataacaactgttatcttgccactgcattgtta

[0228] acactccaacaaatagagttgaagtttaatccacctgctctacaagatgcttattacagagcaagggctggtgaagctgctaacttttgtgcacttatcttagcctactgtaataagacagtaggtgagttaggtgatgttagagaa

[0229] acaatgagttacttgtttcaacatgccaatttagattcttgcaaaagagtcttgaacgtggtgtgtaaaacttgtggacaacagcagacaacccttaagggtgtagaagctgttatgtacatgggcacactttcttatgaacaatttaa

[0230] gaaaggtgttcagataccttgtacgtgtggtaaacaagctacaaaatatctagtacaacaggagtcaccttttgttatgatgtcagcaccacctgctcagtatgaacttaagcatggtacatttacttgtgctagtgagtacactggt

[0231] aattaccagtgtggtcactataaacatataacttctaaagaaactttgtattgcatagacggtgctttacttacaaagtcctcagaatacaaaggtcctattacggatgttttctacaaagaaaacagttacacaacaaccataaaacc

[0232] agttacttataaattggatggtgttgtttgtacagaaattgaccctaagttggacaattattataagaaagacaattcttatttcacagagcaaccaattgatcttgtaccaaaccaaccatatccaaacgcaagcttcgataattttaa

[0233] gtttgtatgtgataatatcaaatttgctgatgatttaaaccagttaactggttataagaaacctgcttcaagagagcttaaagttacatttttccctgacttaaatggtgatgtggtggctattgattataaacactacacaccctcttttaa

[0234] gaaaggagctaaattgttacataaacctattgtttggcatgttaacaatgcaactaataaagccacgtataaaccaaatacctggtgtatacgttgtctttggagcacaaaaccagttgaaacatcaaattcgtttgatgtactgaag

[0235] tcagaggacgcgcagggaatggataatcttgcctgcgaagatctaaaaccagtctctgaagaagtagtggaaaatcctaccatacagaaagacgttcttgagtgtaatgtgaaaactaccgaagttgtaggagacattatactt

[0236] aaaccagcaaataatagtttaaaaattacagaagaggttggccacacagatctaatggctgcttatgtagacaattctagtcttactattaagaaacctaatgaattatctagagtattaggtttgaaaacccttgctactcatggttta

[0237] gctgctgttaatagtgtcccttgggatactatagctaattatgctaagccttttcttaacaaagttgttagtacaactactaacatagttacacggtgtttaaaccgtgtttgtactaattatatgccttatttctttactttattgctacaattgt

[0238] gtacttttactagaagtacaaattctagaattaaagcatctatgccgactactatagcaaagaatactgttaagagtgtcggtaaattttgtctagaggcttcatttaattatttgaagtcacctaatttttctaaactgataaatattataat

[0239] ttggtttttactattaagtgtttgcctaggttctttaatctactcaaccgctgctttaggtgttttaatgtctaatttaggcatgccttcttactgtactggttacagagaaggctatttgaactctactaatgtcactattgcaacctactgtac

[0240] tggttctataccttgtagtgtttgtcttagtggtttagattctttagacacctatccttctttagaaactatacaaattaccatttcatcttttaaatgggatttaactgcttttggcttagttgcagagtggtttttggcatatattcttttcactag

[0241] gtttttctatgtacttggattggctgcaatcatgcaattgtttttcagctattttgcagtacattttattagtaattcttggcttatgtggttaataattaatcttgtacaaatggccccgatttcagctatggttagaatgtacatcttctttgcat

[0242] cattttattatgtatggaaaagttatgtgcatgttgtagacggttgtaattcatcaacttgtatgatgtgttacaaacgtaatagagcaacaagagtcgaatgtacaactattgttaatggtgttagaaggtccttttatgtctatgctaatg

[0243] gaggtaaaggcttttgcaaactacacaattggaattgtgttaattgtgatacattctgtgctggtagtacatttattagtgatgaagttgcgagagacttgtcactacagtttaaaagaccaataaatcctactgaccagtcttcttacat

[0244] cgttgatagtgttacagtgaagaatggttccatccatctttactttgataaagctggtcaaaagacttatgaaagacattctctctctcattttgttaacttagacaacctgagagctaataacactaaaggttcattgcctattaatgtta

[0245] tagtttttgatggtaaatcaaaatgtgaagaatcatctgcaaaatcagcgtctgtttactacagtcagcttatgtgtcaacctatactgttactagatcaggcattagtgtctgatgttggtgatagtgcggaagttgcagttaaaatgtt

[0246] tgatgcttacgttaatacgttttcatcaacttttaacgtaccaatggaaaaactcaaaacactagttgcaactgcagaagctgaacttgcaaaaatgtgtccttagacaatgtcttatctacttttatttcagcagctcggcaagggtt

[0247] tgttgattcagatgtagaaactaaagatgttgttgaatgtcttaaattgtcacatcaatctgacatagaagttactggcgatagttgtaataactatatgctcacctataacaaagttgaaaacatgacaccccgtgaccttggtgcttg

[0248] tattgactgtagtgcgcgtcatattaatgcgcaggtagcaaaaagtcacaacattgctttgatatggaacgttaaagatttcatgtcattgtctgaacaactacgaaaacaaatacgtagtgctgctaaaaagaataacttacctttta

[0249] agttgacatgtgcaactactagacaagttgttaatgttgtaacaacaaagatagcacttaagggtggtaaaaattgttaataattggttgaagcagttaattaaagttacacttgtgttcctttttgttgctgctattttctatttaataacacc

[0250] tgttcatgtcatgtctaaacatactgacttttcaagtgaaatcataggatacaaggctattgatggtggtgtcactcgtgacatagcatctacagatacttgttttgctaacaaacatgctgattttgacacatggtttagccagcgtgg

[0251] tggtagttatactaatgacaaagcttgcccattgattgctgcagtcataacaagagaagtgggttttgtcgtgcctggtttgcctggcacgatattacgcacaactaatggtgactttttgcatttcttacctagagtttttagtgcagtt

[0252] ggtaacatctgttacacaccatcaaaacttatagagtacactgactttgcaacatcagcttgtgttttggctgctgaatgtacaatttttaaagatgcttctggtaagccagtaccatattgttatgataccaatgtactagaaggttctg

[0253] ttgcttatgaaagtttacgccctgacacacgttatgtgctcatggatggctctattattcaatttcctaacacctaccttgaaggttctgttagagtggtaacaacttttgattctgagtactgtaggcacggcacttgtgaaagatcag

[0254] aagctggtgtttgtgtatctactagtggtagatgggtacttaacaatgattattacagatctttaccaggagttttctgtggtgtagatgctgtaaatttatttactaatatgtttacaccactaattcaacctattggtgctttggacatatc

[0255] agcatctatagtagctggtggtattgtggctatcgtagtaacatgccttgcctactattttatgaggtttagaagagcttttggtgaatacagtcatgtagttgcctttaatactttactattccttatgtcattcattgtactctgtttaacac

[0256] cagtttactcattcttacctggtgtttattctgttatttacttgtacttgacattttatcttactaatgatgtttcttttttagcacatattcagtggatggttatgttcacacctttagtacctttctggataacaattgcttatatcatttgtatttcca

[0257] caaagcatttctattggttctttagtaattacctaaagagacgtgtagtctttaatggtgtttcctttagtacttttgaagaagctgcgctgtgcacctttttgttaaataaagaaatgtatctaaagttgcgtagtgatgtgctattacctctt

[0258] acgcaatataatagatacttagctctttataataagtacaagtattttagtggagcaatggatacaactagctacagagaagctgcttgttgtcatctcgcaaaggctctcaatgacttcagtaactcaggttctgatgttctttaccaa

[0259] ccaccacaaatctctatcacctcagctgttttgcagagtggttttagaaaaatggcattcccatctggtaaagttgagggttgtatggtacaagtaacttgtggtacaactacacttaacggtctttggcttgatgacgtagtttactgt

[0260] ccaagacatgtgatctgcacctctgaagatatgcttaaccctaattatgaagatttactcattcgtaagtctaatcataatttcttggtacaggctggtaatgttcaactcagggttattggacattctatgcaaaattgtgtacttaagct

[0261] taaggttgatacagccaatcctaagacacctaagtataagtttgttcgtattcaaccaggacagactttttcagtgttagcttgttacaatggttcaccatctggtgtttaccaatgtgctatgagacacaatttcactattaagggttca

[0262] ttccttaaatggttcatgtggtagtgttggttttaacatagattatgactgtgtctctttttgttacatgcaccatatggaattaccaactggagttcatgctggcacagacttagaaggtaacttttatggaccttttgttgacaggcaaaca

[0263] gcacaagcagctggtacggacacaactattacagttaatgttttagcttggttgtacgctgctgttataaatggagacaggtggtttctcaatcgatttaccacaactcttaatgactttaaccttgtggctatgaagtacaattatgaa

[0264] cctctaacacaagaccatgttgacatactaggacctctttctgctcaaactggaattgccgttttagatatgtgtgcttcattaaaagaattactgcaaaatggtatgaatggacgtaccatattgggtagtgctttattagaagatgaa

[0265] tttacaccttttgatgttgttagacaatgctcaggtgttactttccaaagtgcagtgaaaagaacaatcaagggtacacaccactggttgttactcacaattttgacttcacttttagttttagtccagagtactcaatggtctttgttctttt

[0266] ttttgtatgaaaatgcctttttaccttttgctatgggtattattgctatgtctgcttttgcaatgatgtttgtcaaacataagcatgcatttctctgtttgtttttgttaccttctcttgccactgtagcttattttaatatggtctatatgcctgctagt

[0267] tgggtgatgcgtattatgacatggttggatatggttgatactagtttgaagctaaaagactgtgttatgtatgcatcagctgtagtgttactaatccttatgacagcaagaactgtgtatgatgatggtgctaggagagtgtggacact

[0268] tatgaatgtcttgacactcgtttataaagtttattatggtaatgctttagatcaagccatttccatgtgggctcttataatctctgttacttctaactactcaggtgtagttacaactgtcatgtttttggccagaggtattgtttttatgtgtgtt

[0269] gagtattgccctattttcttcataactggtaatacacttcagtgtataatgctagtttattgtttcttaggctatttttgtacttgttactttggcctcttttgtttactcaaccgctactttagactgactcttggtgtttatgattacttagtttctac

[0270] acaggagtttagatatatgaattcacagggactactcccacccaagaatagcatagatgccttcaaactcaacattaaattgttgggtgttggtggcaaaccttgtatcaaagtagccactgtacagtctaaaatgtcagatgtaaa

[0271] gtgcacatcagtagtcttactctcagttttgcaacaactcagagtagaatcatcatctaaattgtgggctcaatgtgtccagttacacaatgacattctcttagctaaagatactactgaagcctttgaaaaaatggtttcactactttct

[0272] gttttgctttccatgcagggtgctgtagacataaacaagctttgtgaagaaatgctggacaacagggcaaccttacaagctatagcctcagagtttagttcccttccatcatatgcagcttttgctactgctcaagaagcttatgaac

[0273] aggctgttgctaatggtgattctgaagttgttcttaaaaagttgaagaagtctttgaatgtggctaaatctgaatttgaccgtgatgcagccatgcaacgtaagttggaaaagatggctgatcaagctatgacccaaatgtataaac

[0274] aagctagatctgaggacaagagggcaaaagttactagtgctatgcagacaatgcttttcactatgcttagaaagttggataatgatgcactcaacaacattatcaacaatgcaagagatggttgtgttcccttgaacataatacctc

[0275] ttacaacagcagccaaactaatggttgtcataccagactataacacatataaaaatacgtgtgatggtacaacatttacttatgcatcagcattgtgggaaatccaacaggttgtagatgcagatagtaaaattgttcaacttagtga

[0276] aattagtatggacaattcacctaatttagcatggcctcttattgtaacagctttaagggccaattctgctgtcaaattacagaataatgagcttagtcctgttgcactacgacagatgtcttgtgctgccggtactacacaaactgcttg

[0277] cactgatgacaatgcgttagcttactacaacacaacaaagggaggtaggtttgtacttgcactgttatccgatttacaggatttgaaatgggctagattccctaagagtgatggaactggtactatttatacagaactggaaccacc

[0278] ttgtaggtttgttacagacacacctaaaggtcctaaagtgaagtatttatactttattaaaggattaaacaacctaaatagaggtatggtacttggtagtttagctgccacagtacgtctacaagctggtaatgcaacagaagtgcct

[0279] gccaattcaactgtattatctttctgtgcttttgctgtagatgctgctaaagcttacaaagattatctagctagtgggggacaaccaatcactaattgtgttaagatgttgtgtacacacactggtactggtcaggcaataacagttaca

[0280] ccggaagccaatatggatcaagaatcctttggtggtgcatcgtgttgtctgtactgccgttgccacatagatcatccaaatcctaaaggattttgtgacttaaaaggtaagtatgtacaaatacctacaacttgtgctaatgaccctg

[0281] tgggttttacacttaaaaacacagtctgtaccgtctgcggtatgtggaaaggttatggctgtagttgtgatcaactccgcgaacccatgcttcagtcagctgatgcacaatcgtttttaaacgggtttgcggtgtaagtgcagcccg

[0282] tcttacaccgtgcggcacaggcactagtactgatgtcgtatacagggcttttgacatctacaatgataaagtagctggttttgctaaattcctaaaaactaattgttgtcgcttccaagaaaaggacgaagatgacaatttaattgatt

[0283] cttactttgtagttaagagacacactttctctaactaccaacatgaagaaacaatttataatttacttaaggattgtccagctgttgctaaacatgacttctttaagtttagaatagacggtgacatggtaccacatatatcacgtcaacg

[0284] tcttactaaatacacaatggcagacctcgtctatgctttaaggcattttgatgaaggtaattgtgacacattaaaagaaatacttgtcacatacaattgttgtgatgatgattatttcaataaaaaggactggtatgattttgtagaaaac

[0285] ccagatatattacgcgtatacgccaacttaggtgaacgtgtacgccaagctttgttaaaaacagtacaattctgtgatgccatgcgaaatgctggtattgttggtgtactgacattagataatcaagatctcaatggtaactggtatg

[0286] atttcggtgatttcatacaaaccacgccaggtagtggagttcctgttgtagattcttattattcattgttaatgcctatattaaccttgaccagggctttaactgcagagtcacatgttgacactgacttaacaaagccttacattaagtg

[0287] ggatttgttaaaatatgacttcacggaagagaggttaaaactctttgaccgttattttaaatattgggatcagacataccacccaaattgtgttaactgtttggatgacagatgcattctgcattgtgcaaactttaatgttttattctctac

[0288] agtgttcccacttacaagttttggaccactagtgagaaaaatatttgttgatggtgttccatttgtagtttcaactggataccacttcagagagctaggtgttgtacataatcaggatgtaaacttacatagctctagacttagttttaag

[0289] gaattacttgtgtatgctgctgaccctgctatgcacgctgcttctggtaatctattactagataaacgcactacgtgcttttcagtagctgcacttactaacaatgttgcttttcaaactgtcaaacccggtaattttaacaaagacttcta

[0290] tgactttgctgtgtctaagggtttctttaaggaaggaagttctgttgaattaaaacacttcttctttgctcaggatggtaatgctgctatcagcgattatgactactatcgttataatctaccaacaatgtgtgatatcagacaactactatt

[0291] tgtagttgaagttgttgataagtactttgattgttacgatggtggctgtattaatgctaaccaagtcatcgtcaacaacctagacaaatcagctggttttccatttaataaatggggtaaggctagactttattatgattcaatgagttgtg

[0292] aggatcaagatgcacttttcgcatatacaaaacgtaatgtcatccctactataactcaaatgaatcttaagtatgccattagtgcaaagaatagagctcgcaccgtagctggtgtctctatctgtagtactatgaccaatagacagttt

[0293] catcaaaaattattgaaatcaatagccgccactagaggagctactgtagtaattggaacaagcaaattctatggtggttggcacaacatgttaaaaactgtttatagtgatgtagaaaaccctcaccttatgggttgggattatccta

[0294] aatgtgatagagccatgcctaacatgcttagaattatggcctcacttgttcttgctcgcaaacatacaacgtgttgtagcttgtcacaccgtttctatagattagctaatgagtgtgctcaagtattgagtgaaatggtcatgtgtggc

[0295] ggttcactatatgttaaaccaggtggaacctcatcaggagatgccacaactgcttatgctaatagtgtttttaacatttgtcaagctgtcacggccaatgttaatgcacttttatctactgatggtaacaaaattgccgataagtatgtc

[0296] cgcaatttacaacacagactttatgagtgtctctatagaaatagagatgttgacacagactttgtgaatgagttttacgcatatttgcgtaaacatttctcaatgatgatactttctgacgatgctgttgtgtgtttcaatagcacttatgc

[0297] atctcaaggtctagtggctagcataaagaactttaagtcagttctttattatcaaaacaatgtttttatgtctgaagcaaaatgttggactgagactgaccttactaaaggacctcatgaattttgctctcaacatacaatgctagttaaa

[0298] cagggtgatgattatgtgtgtaccttccttacccagatccatcaagaatcctaggggccggctgttttgtagatgatatcgtaaaaacagatggtacacttatgattgaacggttcgtgtctttagctatagatgcttacccacttactaa

[0299] acatcctaatcaggagtatgctgatgtctttcatttgtacttacaatacataagaaagctacatgatgagttaacaggacacatgttagacatgtattctgttatgcttactaatgataacacttcaaggtattgggaacctgagttttat

[0300] gaggctatgtacacaccgcatacagtcttacaggctgttggggcttgtgttctttgcaattcacagacttcattaagatgtggtgcttgcatacgtagaccattcttatgttgtaaatgctgttacgaccatgtcatatcaacatcacat

[0301] aaattagtcttgtctgttaatccgtatgtttgcaatgctccaggttgtgatgtcacagatgtgactcaactttacttaggaggtatgagctattattgtaaatcacataaaaccacccattagttttccattgtgtgctaatggacaagttttt

[0302] ggtttatataaaatacatgtgttggtagcgataatgttactgactttaatgcaattgcaacatgtgactggacaaatgctggtgattacattttagctaacacctgtactgaaagactcaagctttttgcagcagaaacgctcaaagc

[0303] tactgaggagacatttaaactgtcttatggtattgctactgtacgtgaagtgctgtctgacagagaattacatctttcatgggaagttggtaaaacctagaccaccacttaaccgaaattatgtctttactggttatcgtgtaactaaaaaa

[0304] footaagtaacaataggagagtacacctttgaaaaaggtgactatggtgatgctgttgtttaccgaggtacaacaacttacaaattaaatgttggtgattttgtgctgacatcacatacatacagtaatgccattaagtgcacctaca

[0305] ctagtgccacaagagcactatgttagaattactggcttatacccaacactcaatatctcagatgagttttctagcaatgttgcaaattatcaaaggttggtatgcaaaagtattctacactccagggaccacctggtactggtaaga

[0306] gtcattttgctattggcctagctctctactacccttctgctcgcatagtgtatacagcttgctctcatgccgctgttgatgcactatgtgagaaggcattaaaatatttgcctatagataaatgtagtagaattatacctgcacgtgctcg

[0307] tgtagagtgttttgataaattcaaagtgaattcaacattagaacagtatgtcttttgtactgtaaatgcattgcctgagacgacagcagatatagttgtctttgatgaaatttcaatggccacaaattatgatttgagtgttgtcaatgcca

[0308] gattatgtgctaagcactatgtgtacattggcgaccctgctcaattacctgcaccacgcacattgctaactaagggcacactagaaccagaatatttcaattcagtgtgtagacttatgaaaactataggtccagacatgttcctcg

[0309] gaacttgtcggcgttgtcctgctgaaattgttgacactgtgagtgctttggtttatgataataagcttaaagcacataaagacaaatcagctcaatgctttaaaatgttttataagggtgttatcacgcatgatgtttcatctgcaattaa

[0310] caggccacaaataggcgtggtaagagaattccttacacgtaaccctgcttggagaaaagctgtctttatttcaccttataattcacagaatgctgtagcctcaaagattttgggactaccaactcaaactgttgattcatcacaggg

[0311] ctcagaatatgactatgtcatattcactcaaaccactgaaacagctcactcttgtaatgtaaacagatttaatgttgctattaccagagcaaaagtaggcatactttgcataatgtctgatagagacctttatgacaagttgcaatttac

[0312] aagtcttgaaattccacgtaggaatgtggcaactttacaagctgaaaatgtaacaggactctttaaagattgtagtaaggtaatcactgggttacatcctacacaggcacctacacacctcagtgttgacactaaattcaaaactga

[0313] aggtttatgtgttgacgtacctggcatacctaaggacatgacctatagaagactcatctctatgatgggttttaaaatgaattatcaagttaatggttaccctaacatgtttatcacccgcgaagaagctataagacatgtacgtgcat

[0314] ggattggcttcgatgtcgaggggtgtcatgctactagagaagctgttggtaccaaatttacctttacagctaggtttttctacaggtgttaacctagttgctgtacctacaggttatgttgatacacctaataatacagatttttccagagt

[0315] tagtgctaaaccaccgcctggagatcaatttaaacacctcataccacttatgtacaaaggacttccttggaatgtagtgcgtataaagattgtacaaatgttaagtgacacacttaaaatctctctgacagagtcgtatttgtcttat

[0316] gggcacatggctttgagttgacatctatgaagtattttgtgaaaataggacctgagcgcacctgttgtctatgtgatagacgtgccacatgcttttccactgcttcagacacttatgcctgttggcatcattctattggatttgattacgt

[0317] ctataatccgtttatgattgatgttcaacaatggggttttacaggtaacctacaaagcaaccatgatctgtattgtcaagtccatggtaatgcacatgtagctagttgtgatgcaatcatgactaggtgtctagctgtccacgagtgctt

[0318] tgttaagcgtgttgactggactattgaatatcctataattggtgatgaactgaagattaatgcggcttgtagaaaggttcaacacatggttgttaaagctgcattattagcagacaaattcccagttcttcacgacattggtaacccta

[0319] aagctattaagtgtgtacctcaagctgatgtagaatggaagttctatgatgcacagccttgtagtgacaaagcttataaaatagaagaattattctattcttatgccacacattctgacaaattcacagatggtgtatgcctattttgga

[0320] attgcaatgtcgatagatatcctgctaattccattgtttgtagatttgacactagagtgctatctaaccttaacttgcctggttgtgatggtggcagtttgtatgtaaataaacatgcattccacacaccagcttttgataaaagtgcttttg

[0321] ttaatttaaaacaattaccatttttctattactctgacagtccatgtgagtctcatggaaaacaagtagtgtcagatatagattatgtaccactaaagtctgctacgtgtataacacgttgcaatttaggtggtgctgtctgtagacatcat

[0322] gctaatgagtacagattgtatctcgatgcttataacatgatgatctcagctggctttagcttgtgggtttacaaacaatttgatacttataacctctggaacacttttacaaagacttcagagtttagaaaatgtggcttttaatgttgtaaat

[0323] aagggacactttgatggacaacagggtgaagtaccagtttctatcattaataacactgtttacacaaaagttgatggtgttgatgtagaattgtttgaaaataaaacaacattacctgttaatgtagcatttgagctttgggctaagcg

[0324] caacattaaaccagtaccagaggtgaaaatactcaataatttgggtgtggacattgctgctaatactgtgatctgggactacaaaagagatgctccagcacatatatctactattggtgtttgttctatgactgacatagccaagaa

[0325] accaattgaaacgatttgtgcaccactcactgtcttttttgatggtagagttgatggtcaagtagacttatttagaaatgcccgtaatggtgttcttattacagagggtagtgttaaaggtttacaaccatctgtaggtcccaaacaagc

[0326] tagtcttaatggagtcacattaattggagaagccgtaaaaacacagttcaattattataagaaagttgatggtgttgtccaacaattacctgaaacttactttactcagagtagaaatttacaagaatttaaacccaggagtcaaatg

[0327] gaaattgatttcttagaattagctatggatgaattcattgaacggtataaattagaaggctatgccttcgaacatatcgtttatggagattttagtcatagtcagttaggtggtttacatctactgattggactagctaaacgttttaagga

[0328] atcaccttttgaattagaagattttattcctatggacagtacagttaaaaactatttcataacagatgcgcaaacaggttcatctaagtgtgtgtgttctgttattgatttattacttgatgattttgttgaaataataaaatcccaagatttat

[0329] ctgtagtttctaaggttgtcaaagtgactattgactatacagaaatttcatttatgctttggtgtaaagatggccatgtagaaacattttacccaaattacaatctagtcaagcgtggcaaccgggtgttgctatgcctaatctttacaa

[0330] aatgcaaagaatgctattagaaaagtgtgaccttcaaaattatggtgatagtgcaacattacctaaaggcataatgatgaatgtcgcaaatatactcaactgtgtcaatatttaaacacattaacattagctgtaccctataatatga

[0331] gagttatacattttggtgctggttctgataaaggagttgcaccaggtacagctgttttaagacagtggttgcctacgggtacgctgcttgtcgattcagatcttaatgactttgtctctgatgcagattcaactttgattggtgattgtgc

[0332] aactgtacatacagctaataaatgggatctcattattagtgatatgtacgaccctaagactaaaaatgttacaaaagaaaatgactctaaagagggttttttcacttacatttgtgggtttatacaacaaaagctagctcttggaggttc

[0333] cgtggctataaagataacagaacattcttggaatgctgatctttataagctcatgggacacttcgcatggtggacagcctttgttactaatgtgaatgcgtcatcatctgaagcatttttaattggatgtaattatcttggcaaaccacg

[0334] cgaacaaatagatggttatgtcatgcatgcaaattacatattttggaggaatacaaatccaattcagttgtcttcctattctttatttgacatgagtaaatttccccttaaattaaggggtactgctgttatgtctttaaaagaaggtcaaat

[0335] caatgatatgattttatctcttcttagtaaaggtagacttataattagagaaaacaacagagttgttatttctagtgatgttcttgttaacaactaaacgaacaatgtttgtttttcttgttttattgccactagtctctagtcagtgtgttaatct

[0336] tataaccagaactcaatcatacactaattctttcacacgtggtgtttattaccctgacaaagttttcagatcctcagttttacattcaactcaggacttgttcttacctttcttttccaatgttacttggttccatgctatctctgggaccaatg

[0337] gtactaagaggtttgataaaccctgtcctaccatttaaatgatggtgtttattttgcttccactgagaagtctaacataataagaggctggatttttggtactactttagattcgaagaacccagtccctacttattgttaataacgctactaat

[0338] gttgttattaaagtctgtgaatttcaattttgtaatgatccatttttggatgtttattaccacaaaaacaacaaaagttggatggaaagtgagttcagagtttattctagtgcgaataattgcacttttgaatatgtctctcagccttttcttat

[0339] ggaccttgaaggaaaacagggtaatttcaaaaatcttagggaatttgtgtttaagaatattgatggttattttaaaatatattctaagcacacgcctattaatttagggcgtgatctccctcagggtttttcggctttagaaccattggta

[0340] gatttgccaataggtattaacatcactaggtttcaaactttacttgctttacatagaagttattgactcctggtgattcttcttcaggttggacagctggtgctgcagcttattatgtgggttatcttcaacctaggacttttctattaaaata

[0341] taatgaaaatggaaccattacagatgctgtagactgtgcacttgaccctctctcagaaacaaagtgtacgttgaaatccttcactgtagaaaaaggaatctatcaaacttctaactttagagtccaaccaacagaatctattgttaga

[0342] tttcctaatattacaaacttgtgcccttttgatgaagtttttaacgccaccagatttgcatctgtttatgcttggaacaggaagagaatcagcaactgtgttgctgattattctgtcctatataatttcgcaccatttttcgcttttaagtgttat

[0343] ggagtgtctcctactaaattaaatgatctctgctttactaatgtctatgcagattcatttgtaattagaggtaatgaagtcagccaaatcgctccagggcaaactggaaatattgctgattataattataaattaccagatgattttacag

[0344] gctgcgttatagcttggaattctaacaagcttgattctaaggttggtggtaattataattaccggtatagattgtttaggaagtctaatctcaaaccttttgagagagatatttcaactgaaatctatcaggccggtaacaaaccttgtaa

[0345] tggtgttgcaggtgttaattgttactttcctttacaatcatatggtttccgacccacttatggtgttggtcaccaaccatacagagtagtagtactttcttttgaacttctacatgcaccagcaactgtttgtggacctaaaaagtctacta

[0346] atttggttaaaaacaaatgtgtcaatttcaacttcaatggtttaacaggcacaggtgttcttactgagtctaacaaaaagtttctgcctttccaacaatttggcagagacattgctgacactactgatgctgtccgtgatccacagaca

[0347] cttgagattcttgacattacaccatgttcttttggtggtgtcagtgttataacaccaggaacaaatacttctaaccaggttgctgttctttatcagggtgttaactgcacagaagtccctgttgctattcatgcagatcaacttactcctac

[0348] ttggcgtgtttattctacaggttctaatgtttttcaaacacgtgcaggctgtttaataggggctgaatatgtcaacaactcatatgagtgtgacatacccattggtgcaggtatatgcgctagttatcagactcagactaagtctcatcg

[0349] gcgggcacgtagtgtagctagtcaatccatcattgcctacactatgtcacttggtgcagaaaattcagttgcttactctaataactctattgccatacccacaaattttactattagtgttaccacagaaattctaccagtgtctatgac

[0350] caagacatcagtagattgtacaatgtacatttgtggtgattcaactgaatgcagcaatcttttgttgcaatatggcagtttttgtacacaattaaaacgtgctttaactggaatagctgttgaacaagacaaaaacacccaagaagttt

[0351] ttgcacaagtcaaacaaatttacaaaacaccaccaattaaatattttggtggttttaatttttcacaaatattaccagatccatcaaaaccaagcaagaggtcatttattgaagatctacttttcaacaaagtgacacttgcagatgctg

[0352] gcttcatcaaacaatatggtgattgccttggtgatattgctgctagagacctcatttgtgcacaaaagtttaacggccttactgttttgccacctttgctcacagatgaaatgattgctcaatacacttctgcactgttagcgggtacaa

[0353] tcacttctggttggacctttggtgcaggtgctgcattacaaataccatttgctatgcaaatggcttataggtttaatggtattggagttacacagaatgttctctatgagaaccaaaaattgattgccaaccaatttaatagtgctattgg

[0354] caaaattcaagactcactttcttccacagcaagtgcacttggaaaacttcaagatgtggtcaaccataatgcacaagctttaaacacgcttgttaaacaacttagctccaaatttggtgcaatttcaagtgttttaaatgatatcctttc

[0355] acgtcttgacaaagttgaggctgaagtgcaaattgataggttgatcacaggcagacttcaaagtttgcagacatatgtgactcaacaattaattagagctgcagaaatcagagcttctgctaatcttgctgctactaaaatgtcaga

[0356] gtgtgtacttggacaatcaaaaagagttgatttttgtggaaagggctatcatcttatgtccttccctcagtcagcacctcatggtgtagtcttcttgcatgtgacttatgtccctgcacaaagaaaagaacttcacaactgctcctgccat

[0357] ttgtcatgatggaaaagcacactttcctcgtgaaggtgtctttgtttcaaatggcacacactggtttgtaacacaaaggaatttttatgaaccacaaatcattactacagacaacacatttgtgtctggtaactgtgatgttgtaatagg

[0358] aattgtcaacaacacagtttatgatcctttgcaacctgaattagattcattcaaggaggagttagataaatattttaagaatcatacatcaccagatgttgatttaggtgacatctctggcattaatgcttcagttgtaaacattcaaaaa

[0359] gaaattgaccgcctcaatgaggttgccaagaatttaaatgaatctctcatcgatctccaagaacttggaaagtatgagcagtatataaaatggccatggtacatttggctaggttttatagctggcttgattgccatagtaatggtga

[0360] caattatgctttgctgtatgaccagttgctgtagttgtctcaaagggctgttgttcttgtggatcctgctgcaaatttgatgaagacgactctgagccagtgctcaaaggagtcaaattacattacacataaacgaacttatggatttgttt

[0361] atgagaatcttcacaattggaactgtaactttgaagcaaggtgaaatcaaggatgctactccttcagattttgttcgcgctactgcaacgataccgatacaagcctcactccctttcggatggcttattgttggcgttgcacttcttgc

[0362] tgtttttcagagcgcttccaaaatcataactctcaaaaagagatggcaactagcactctccaagggtgttcactttgtttgcaacttgctgttgttgtttgtaacagtttactcacaccttttgctcgttgctgctggccttgaagcccctt

[0363] ttctctatctttatgctttagtctacttcttgcagagtataaactttgtaagaataatgaggctttggctttgctggaaatgccgttccaaaaacccattactttatgatgccaactattttctttgctggcatactaattgttacgactatt

[0364] gtataccttacaatagtgtaacttcttcaattgtcattacttcaggtgatggcacaacaagtcctatttctgaacatgactaccagattggtggttatactgaaaaatgggaatctggagtaaaagactgtgttgtattacacagttactt

[0365] cacttcagactattaccagctgtactcaactcaattgagtacagacattggtgttgaacatgttaccttcttcatctacaataaaattgttgatgagcctgaagaacatgtccaaattcacacaatcgacggttcatccggagttgtta

[0366] atccagtaatggaaccaatttatgatgaaccgacgacgactactagcgtgcctttgtaagcacaagctgatgagtacgaacttatgtactcattcgtttcggaagagataggtacgttaaatagttaatagcgtacttctttttcttgctt

[0367] tcgtggtattcttgctagttacactagccatccttactgcgcttcgattgtgtgcgtactgctgcaatattgttaacgtgagtcttgtaaaaccttctttttacgtttactctcgtgttaaaaatctgaattcttctagagttcctgatcttctgg

[0368] tctaaacgaactaaatattatattagtttttctgtttggaactttaattttagccatggcaaattccaacggtactattaccgttgaagagcttaaaaagctccttgaagaatggaacctagtaataggtttcctattccttacatggatttgt

[0369] cttctacaatttgcctatgccaacaggaataggtttttgtatataattaagttaattttcctctggctgttatggccagtaactttaacttgttttgtgcttgctgctgtttacagaataaattggatcaccggtggaattgctatcgcaatgg

[0370] cttgtcttgtaggcttgatgtggctcagctacttcattgcttctttcagactgtttgcgcgtacgcgttccatgtggtcattcaatccagaaactaacattcttctcaacgtgccactccatggcactattctgaccagaccgcttctaga

[0371] aagtgaactcgtaatcggagctgtgatccttcgtggacatcttcgtattgctggacaccatctaggacgctgtgacatcaaggacctgcctaaagaaatcactgttgctacatcacgaacgctttcttattacaaattgggagcttc

[0372] gcagcgtgtagcaggtgactcaggttttgctgcatacagtcgctacaggattggcaactataaattaaacacagaccattccagtagcagtgacaatattgctttgcttgtacagtaagtgacaacagatgtttcatctcgttgactt

[0373] tcaggttactatagcagagatattactaattattatgaggacttttaaagtttccatttggaatcttgattacatcataaacctcataattaaaaatttatctaagtcactaactgagaataaatattctcaattagatgaagagcaaccaat

[0374] ggagattgattaaacgaacatgaaaattattcttttcttggcactgataacactcgctacttgtgagctttatcactaccaagagtgtgttagaggtacaacagtacttttaaaagaaccttgctcttctggaacatacgagggcaatt

[0375] caccatttcatcctctagctgataacaaatttgcactgacttgctttagcactcaatttgcttttgcttgtcctgacggcgtaaaacacgtctatcagttacgtgccagatcagtttcacctaaactgttcatcagacaagaggaagttc

[0376] aagaactttactctccaatttttcttattgttgcggcaatagtgtttataacactttgcttcacactcaaaagaaagacagaatgattgaactttcattaattgacttctatttgtgctttttagcctttctgttattccttgttttaattatgcttatt

[0377] atcttttggttctcacttgaactgcaagatcataatgaaacttgtcacgcctaaatgaacatgaaatttcttgttttcttaggaatcatcacaactgtagctgcatttcaccaagaatgtagtttacagtcatgtactcaacatcaaccata

[0378] tgtagttgatgacccgtgtcctattcacttctattctaaatggtatattagagtaggagctagaaaaatcagcacctttaattgaattgtgcgtggatgaggctggttctaaatcacccattcagtacatcgatatcggtaattatacagtt

[0379] tcctgtttaccttttacaattaattgccaggaacctaaattgggtagtcttgtagtgcgttgttcgttctatgaagactttttagagtatcatgacgttcgtgttgttttagatttcatctaaacgaacaaacttaaatggtcttcacactcga

[0380] agatttcgttggggactggcgacagacagccggctacaacctggaccaagtccttgaacagggaggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaatggg

[0381] ctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgaccaaatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtgatcctgcactatggcacactggtaatcg

[0382] acggggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaacagggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgac

[0383] ggctccctgctgttccgagtaaccatcaacggagtgaccggctggcggctgtgcgaacgcattctggcgggctctagcggcatgggcgtgatcaagcccgacatgaagatcaagctgcggatggagggcgccgtgaac

[0384] ggccacaaattcgtgatcgagggcgacgggaaaggcaagccctttgagggtaagcagactatggacctgaccgtgatcgagggcgcccccctgcccttcgcttatgacattctcaccaccgtgttcgactacggtaaccgt

[0385] gtcttcgccaagtaccccaaggacatccctgactacttcaagcagaccttccccgagggctactcgtgggagcgaagcatgacatacgaggaccagggaatctgtatcgctacaaacgacatcaccatgatgaagggtgtg

[0386] gacgactgcttcgtgtacaaaatccgcttcgacggggtcaacttccctgctaatggcccggtgatgcagcgcaagaccctaaagtgggagcccagtaccgagaagatgtacgtgcgggacggcgtactgaagggcgatgt

[0387] taatatggcactgctcttggagggaggcggccactaccgctgcgacttcaagaccacctacaaagccaagaaggtggtgcagcttcccgactaccacttcgtggaccaccgcatcgagatcgtgagccacgacaaggact

[0388] acaacaaagtcaagctgtacgagcacgccgaagcccacagcggactaccccgccaggccggctaaactcatgcagaccacacaaggcagatgggctatataaacgttttcgcttttccgtttacgatatatagtctactcttgt

[0389] gcagaatgaattctcgtaactacatagcacaagtagatgtagttaactttaatctcacatagcaatctttaatcagtgtgtaacattagggaggacttgaaagagccaccacattttcaccgaggccacgcggagtacgatcgagt

[0390] gtacagtgaacaatgctagggagagctgcctatatggaagagccctaatgtgtaaaattaattttagtagtgctatccccatgtgattttaatagcttcttaggagaatgacaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa

[0391] gcggccgcgatcgcttgcctgtaacttacacgcgcctcgtatcttttaatgatggaataatttgggaatttactctgtgtttatttatttttatgttttgtatttggattttagaaagtaaataaagaaggtagaagagttacggaatgaag

[0392] aaaaaaaaataaacaaaggtttaaaaaatttcaacaaaaagcgtactttacatatatatttattagacaagaaaagcagattaaatagatatacattcgattaacgataagtaaaatgtaaaatcacaggattttcgtgtgtggtcttct

[0393] acacagacaagatgaaacaattcggcattaatacctgagagcaggaagagcaagataaaaggtagtatttgttggcgatccccctagagtcttttacatcttcggaaaacaaaaactattttttctttaatttctttttttactttctatttt

[0394] taatttatatatttatattaaaaaatttaaattataattatttttatagcacgtgatgaaaaggaccctctccccgcgcgttggccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacg

[0395] caattaatgtgagttagctcactcattaggcaccccaggctttacactttatgcttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagctttaagccagccccgacacccgccaacacccg

[0396] ctgacgcgccctgacgggcttgtctgctcccggcatccgcttacagacaagctgtgaccgtctccgggagctgcatgtgtcagaggttttcaccgtcatcaccgaaacgcgcgagacgaaagggcctcgtgatacgcctatt

[0397] tttataggttaatgtcatgataataatggtttcttagacgtcgtatttttttttttttagagaaaatcctccaatatcaaattaggaatcgtagtttcatgattttctgttacacctaactttttgtgtggtgccctcctccttgtcaatattaatgtt

[0398] aaagtgcaattctttttccttatcacgttgagccattagtatcaatttgcttacctgtattcctttactatcctcctttttctccttcttgataaatgtatgtagattgcgtatatagtttcgtctacccctatgaacatattccatttgtaatttcgtg

[0399] tcgtttctattatgaatttcatttataaagtttatgtacaaatatcataaaaaaagagaatctttctatttcttagcatttttgacgaaatttgctattttgttagagtcttttacaccatttgtctccacacctccgcttacatcaacaccaataa

[0400] cgccatttaaatctaagcgcatcaccaacattttctggcgtcagtccaccagctaacataaaatgtaaactttcggggctctcttgccttccaacccagtcagaaatcgagttccaatccaaaagttcacctgtcccacctgcttctga

[0401] atcaaacaagggaataaacgaatgaggtttctgtgaagctgcactgagtagtatgttgcagtcttttggaaatacgagtcttttaataactggcaaaccgaggaactcttggtattcttgccacgactcatctccatgcagttggact

[0402] atatcaatgccgtaatcattgaccagagccaaaacatcctccttaggttgattacgaaacacgccaaccaagtatttcggagtgcctgaactatttttatatgcttttacaagacttgaaattttccttgcaataaccgggtcaattgtt

[0403] ctctttctattgggcacacatataatacccagcaagtcagcatcggaatctagagcacattctgcggcctctgtgctctgcaagccgcaaactttcaccaatggaccagaactacctgtgaaattaataacagacatactccaag

[0404] ctgcctttgtgtgcttaatcacgtatactcacgtgctcaatagtcaccaatgccctccctcttggccctctccttttcttttttcgaccgaattaattcttaatcggcaaaaaaagaaaagctccggatcaagattgtacgtaaggtgac

[0405] aagctatttttcaataaagaatatcttccactactgccatctggcgtcataactgcaaagtacacatatattacgatgctgttctattaaatgcttcctatattatatatatagtaatgtcgttagctcggtacccggggatcctctagagt

[0406] cgacctgcaggcatgcaagcttgagtattctatagtctcacctaaatagcttggcgtaatcatggtcatagctgtttcctgtgtgaaattgttatccgctcacaattccacacaacatacgagccggaagcataaagtgtaaagcct

[0407] ggggtgcctaatgagtgagctaactcacattaattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccagctgcattaatgaatcggccaacgcgaaccccttgcggccgcccgggccgtcgaccaattc

[0408] tcatgtttgacagcttatcatcgaatttctgccattcatccgcttattatcacttattcaggcgtagcaaccaggcgtttaagggcaccaataactgccttaaaaaaattacgccccgccctgccactcatcgcagtactgttgtaattc

[0409] attaagcattctgccgacatggaagccatcacaaacggcatgatgaacctgaatcgccagcggcatcagcaccttgtcgccttgcgtataatatttgcccatggtgaaaacgggggcgaagaagttgtccatattggccacgtt

[0410] taaatcaaaactggtgaaactcacccagggattggctgagacgaaaaacatattctcaataaaccctttagggaaataggccaggttttcaccgtaacacgccacatcttgcgaatatatgtgtagaaactgccggaaatcgtc

[0411] gtggtattcactccagagcgatgaaaacgtttcagtttgctcatggaaaacggtgtaacaagggtgaacactatcccatatcaccagctcaccgtctttcattgccatacgaaattccggatgagcattcatcaggcgggcaaga

[0412] atgtgaataaaggccggataaaacttgtgcttatttttctttacggtctttaaaaaggccgtaatatccagctgaacggtctggttataggtacattgagcaactgactgaaatgcctcaaaatgttctttacgatgccattgggatata

[0413] tcaacggtggtatatccagtgatttttttctccattttagcttccttagctcctgaaaatctcgataactcaaaaaatacgcccggtagtgatcttatttcattatggtgaaagttggaacctcttacgtgccgatcaacgtctcattttcgc

[0414] caaaagttggcccagggcttcccggtatcaacagggacaccaggatttatttattctgcgaagtgatcttccgtcacaggtatttattcgcgataagctcatggagcggcgtaaccgtcgcacaggaaggacagagaaagcg

[0415] cggatctgggaagtgacggacagaacggtcaggacctggattggggaggcggttgccgccgctgctgctgacggtgtgacgttctctgttccggtcacaccacatacgttccgccattcctatgcgatgcacatgctgtatg

[0416] ccggtataccgctgaaagttctgcaaagcctgatgggacataagtccatcagttcaacggaagtctacacgaaggtttttgcgctggatgtggctgcccggcaccgggtgcagtttgcgatgccggagtctgatgcggttgcg

[0417] atgctgaaacaattatcctgagaataaatgccttggcctttatatggaaatgtggaactgagtggatatgctgtttttgtctgttaaacagagaagctggctgttatccactgagaagcgaacgaaacagtcgggaaaatctcccat

[0418] tatcgtagagatccgcattattaatctcaggagcctgtgtagcgtttataggaagtagtgttctgtcatgatgcctgcaagcggtaacgaaaacgatttgaatatgccttcaggaacaatagaaatcttcgtgcggtgttacgttgaa

[0419] gtggagcggattatgtcagcaatggacagaacaacctaatgaacacagaaccatgatgtggtctgtccttttacagccagtagtgctcgccgcagtcgagcgacagggcgaagccctcgagctggttgccctcgccgctgg

[0420] gctggcggccgtctatggccctgcaaacgcgccagaaacgccgtcgaagccgtgtgcgagacaccgcggccggccgccggcgttgtggatacctcgcggaaaacttggccctcactgacagatgaggggcggacgtt

[0421] gacacttgaggggccgactcacccggcgcggcgttgacagatgaggggcaggctcgatttcggccggcgacgtggagctggccagcctcgcaaatcggcgaaaacgcctgattttacgcgagtttcccacagatgatgt

[0422] ggacaagcctggggataagtgccctgcggtattgacacttgaggggcgcgactactgacagatgaggggcgcgatccttgacacttgaggggcagagtgctgacagatgaggggcgcacctattgacatttgaggggct

[0423] gtccacaggcagaaaatccagcatttgcaagggtttccgcccgtttttcggccaccgctaacctgtcttttaacctgcttttaaaccaatatttataaaccttgtttttaaccagggctgcgccctgtgcgcgtgaccgcgcacgcc

[0424] gaaggggggtgcccccccttctcgaaccctcccggtcgagtgagcgaggaagcaccagggaacagcacttatatattctgcttacacacgatgcctgaaaaaacttcccttggggttatccacttatccacggggatattttta

[0425] taattatttttttatagttttagatcttttttagagcgccttgtagcctttatccatgctggttctagagaaggtgttgtgacaaattgccctttcagtgtgacaaatcaccctcaaatgacagtcctgtctgtgacaaattgccctta

[0426] accctgtgacaaattgccctcagaagaagctgttttttcacaaagttatccctgcttattgactctttttatttagtgtgacaatctaaaaacttgtcacacttcacatggatctgtcatggcggaaacagcggttatcaatcacaagaa

[0427] acgtaaaaatagcccgcgaatcgtccagtcaaacgacctcactgaggcggcatagatctctcccgggatcaaaaacgtatgctgtatctgttcgttgaccagatcagaaaatctgatggcaccctacaggaaacatgacggta

[0428] tctgcgagatccatgttgctaaatatgctgaaatattcggattgacctctgcggaagccagtaaggatatacggcaggcattgaagagtttcgcggggaaggaagtggttttttatcgccctgaagaggatgccggcgatgaaa

[0429] aaggctatgaatcttttccttggtttatcaaacgtgcgcacagtccatccagagggctttacagtgtacatatcaacccatatctcattcccttctttatcgggttacagaaccggtttacgcagtttcggcttagtgaaacaaaagaa

[0430] atcaccaatccgtatgccatgcgtttatacgaatccctgtgtcagtatcgtaagccggatggctcaggcatcgtctctctgaaaatcgactggatcatagagcgttaccagctgcctcaaagttaccagcgtatgcctgacttccg

[0431] ccgccgcttcctgcaggtctgtgttaatgagatcaacagcagaactccaatgcgcctctcatacattgagaaaaagaaaggccgccagacgactcatatcgtattttccttccgcgatatcacttccatgacgacaggatagtct

[0432] gagggttatctgtcacagatttgagggtggttcgtcacatttgttctgacctactgagggtaatttgtcacagttttgctgtttccttcagcctgcatggattttctcatactttttgaactgtaatttttaaggaagccaaatttgagggca

[0433] gtttgtcacagttgatttccttctctttcccttcgtcatgtgacctgatatcgggggttagttcgtcatcattgatgagggttgattatcacagtttattactctgaattggctatccgcgtgtgtacctctacctggagtttttcccacggtg

[0434] gatatttcttcttgcgctgagcgtaagagctatctgacagaacagttcttctttgcttcctcgccagttcgctcgctatgctcggttacacggctgcggcgagcgctagtgataataagtgactgaggtatgtgctcttcttatctcctt

[0435] ttgtagtgttgctcttattttaaacaactttgcggttttttgatgactttgcgattttgttgttgctttgcagtaaattgcaagatttaataaaaaaacgcaaagcaatgattaaaggatgttcagaatgaaactcatggaaacacttaacca

[0436] gtgcataaacgctggtcatgaaatgacgaaggctatcgccattgcacagtttaatgatgacagcccggaagcgaggaaaataacccggcgctggagaataggtgaagcagcggatttagttggggtttcttctcaggctatc

[0437] agagatgccgagaaagcagggcgactaccgcacccggatatggaaattcgaggacgggttgagcaacgtgttggttatacaattgaacaaattaatcatatgcgtgatgtgtttggtacgcgattgcgacgtgctgaagacgt

[0438] atttccaccggtgatcggggttgctgcccataaaggtggcgtttacaaaacctcagtttctgttcatcttgctcaggatctggctctgaaggggctacgtgttttgctcgtggaaggtaacgacccccagggaacagcctcaatgt

[0439] atcacggatgggtaccagatcttcatattcatgcagaagacactctcctgcctttctatcttggggaaaaggacgatgtcacttatgcaataaagcccacttgctggccggggcttgacattattccttcctgtctggctctgcacc

[0440] gtattgaaactgagttaatgggcaaatttgatgaaggtaaactgcccaccgatccacacctgatgctccgactggccattgaaactgttgctcatgactatgatgtcatagttattgacagcgcgcctaacctgggtatcggcacg

[0441] attaatgtcgtatgtgctgctgatgtgctgattgttcccacgcctgctgagttgtttgactacacctccgcactgcagtttttcgatatgcttcgtgatctgctcaagaacgttgatcttaaagggttcgagcctgatgtacgtattttgct

[0442] taccaaatacagcaatagcaatggctctcagtccccgtggatggaggagcaaattcgggatgcctggggaagcatggttctaaaaaatgttgtacgtgaaacggatgaagttggtaaaggtcagatccggatgagaactgttt

[0443] ttgaacaggccattgatcaacgctcttcaactggtgcctggagaaatgctctttctatttgggaacctgtctgcaatgaaattttcgatcgtctgattaaaccacgctgggagattagataatgaagcgtgcgcctgttattccaaaa

[0444] catacgctcaatactcaaccggttgaagatacttcgttatcgacaccagctgccccgatggtggattcgttaattgcgcgcgtaggagtaatggctcgcggtaatgccattactttgcctgtatgtggtcgggatgtgaagtttact

[0445] cttgaagtgctccggggtgatagtgttgagaagacctctcgggtatggtcaggtaatgaacgtgaccaggagctgcttactgaggacgcactggatgatctcatcccttcttttctactgactggtcaacagacaccggcgttcg

[0446] gtcgaagagtatctggtgtcatagaaattgccgatgggagtcgccgtcgtaaagctgctgcacttaccgaaagtgattatcgtgttctggttggcgagctggatgatgagcagatggctgcattatccagattgggtaacgatta

[0447] tcgcccaacaagtgcttatgaacgtggtcagcgttatgcaagccgattgcagaatgaatttgctggaaatatttctgcgctggctgatgcggaaaatatttcacgtaagattattacccgctgtatcaacaccgccaaattgcctaa

[0448] atcagttgttgctcttttttctcaccccggtgaactatctgcccggtcaggtgatgcacttcaaaaagcctttacagataaagaggaattacttaagcagcaggcatctaaccttcatgagcagaaaaaagctggggtgatatttga

[0449] agctgaagaagttatcactcttttaacttctgtgcttaaaacgtcatctgcatcaagaactagtttaagctcacgacatcagtttgctcctggagcgacagtattgtataagggcgataaaatggtgcttaacctggacaggtctcgt

[0450] gttccaactgagtgtatagagaaaattgaggccattcttaaggaacttgaaaagccagcaccctgatgcgaccacgttttagtctacgtttatctgtctttacttaatgtcctttgttacaggccagaaagcataactggcctgaatat

[0451] tctctctgggcccactgttccacttgtatcgtcggtctgataatcagactgggaccacggtcccactcgtatcgtcggtctgattattagtctgggaccacggtcccactcgtatcgtcggtctgattattagtctgggaccacggtc

[0452] ccactcgtatcgtcggtctgataatcagactgggaccacggtcccactcgtatcgtcggtctgattattagtctgggaccatggtcccactcgtatcgtcggtctgattattagtctgggaccacggtcccactcgtatcgtcggtc

[0453] tgattattagtctggaaccacggtcccactcgtatcgtcggtctgattattagtctgggaccacggtcccactcgtatcgtcggtctgattattagtctgggaccacgatcccactcgtgttgtcggtctgattatcggtctgggacc

[0454] acggtcccacttgtattgtcgatcagactatcagcgtgagactacgattccatcaatgcctgtcaagggcaagtattgacatgtcgtcgtaacctgtagaacggagtaacctcggtgtgcggttgtatgcctgctgtggattgctg

[0455] ctgtgtcctgcttatccacaacattttgcgcacggttatgtggacaaaatacctggttacccaggccgtgccggcacgttaaccgggctgcatccgatgcaagtgtgtcgctgtcgacgagctcgcgagctcggacatgaggtt

[0456] gccccgtattcagtgtcgctgatttgtattgtctgaagttgtttttacgttaagttgatgcagatcaattaatacgatacctgcgtcataattgattatttgacgtggtttgatggcctccacgcacgttgtgatatgtagatgataatcatt

[0457] atcactttacgggtcctttccggtgatccgacaggttacggggcggcgacctcgcgggttttcgctatttatgaaaattttccggtttaaggcgtttccgttcttcttcgtcataacttaatgtttttatttaaaataccctctgaaaagaaaggaaacgacaggtgctgaaagcgagctttttggcctctgtcgtttcctttctctgtttttgtccgtggaatgaacaatggaagtccgagctcatcgctaataacttcgtatagcatacattatacgaagttatattcgat。

[0458] On the other hand, an embodiment of the present invention provides a viral replicon system, which includes: a cell that stably expresses the new coronavirus SARS-CoV-2N protein and an expression vector containing the viral replicon described in any of the preceding embodiments or the DNA molecule described in the preceding embodiments.

[0459] It is understandable that, on the expression vector containing the viral replicon, the nucleotide sequence of the viral replicon is a nucleotide sequence in which U is replaced by T.

[0460] In some embodiments, the vector backbone of the expression vector may be: pCC1-BAC vector.

[0461] In some embodiments, the expression vector is located in the cell.

[0462] In some embodiments, the cell is a cell that stably expresses the novel coronavirus SARS-CoV-2 N protein and transmembrane serine protease.

[0463] In some embodiments, the cells can be mammalian cells and cell lines. "Mammalian cells" include cells from any member of the mammalian family, such as human cells, mouse cells, rat cells, monkey cells, hamster cells, etc. For example, the cells can be any one or more of Vero E6 cells, BHK-21-hACE2 cells, CACO-2 cells, and HEK293T-hACE2 cells.

[0464] Most of the replicon systems reported so far do not produce virus-like particles or the titer of the virus-like particles produced is not high. In order to achieve the preparation of high-titer replicons, on the one hand, the present application screens out cells with high expression of N protein through monoclonal screening to produce virus-like particles; on the other hand, during the adsorption of the new coronavirus SARS-CoV-2 to cells, the transmembrane serine protease (TMPRSS2) can activate the SARS-CoV-2S protein and induce the fusion of the virus and cells, promoting infection; the appropriate concentration of trypsin in the culture medium can also promote the virus to infect cells; therefore, the present application stably expresses TMPRSSS2 protein on receptor cells with high expression of N protein, and adds a suitable concentration of trypsin to the culture medium when the replicon infects the cells to prepare a high-titer replicon.

[0465] On the other hand, an embodiment of the present invention provides a method for producing a viral replicon, comprising: culturing a cell stably expressing the novel coronavirus SARS-CoV-2 N protein as described in any of the preceding embodiments.

[0466] In some embodiments, the production method includes culturing cells that stably express the novel coronavirus SARS-CoV-2 N protein and contain an expression vector of the DNA molecule.

[0467] In some embodiments, the culturing comprises: adding trypsin to the culture environment of the cells. Compared with not adding trypsin, adding trypsin can increase the titer of the prepared replicon.

[0468] In some embodiments, the active concentration of trypsin in the culture environment is 1 to 40 μg / ml, specifically any one of 1 μg / ml, 5 μg / ml, 10 μg / ml, 15 μg / ml, 20 μg / ml, 25 μg / ml, 30 μg / ml, 35 μg / ml and 40 μg / ml, or a range between any two of them.

[0469] In some embodiments, the trypsin has an effective concentration of 15 to 25 μg / ml in the culture environment, preferably 20 μg / ml.

[0470] On the other hand, an embodiment of the present invention provides the use of the viral replicon described in any of the preceding embodiments, or the DNA molecule described in any of the preceding embodiments, or the viral replicon system described in any of the preceding embodiments, or the viral replicon generated by the production method described in any of the preceding embodiments in drug screening against the new coronavirus SARS-Cov-2.

[0471] On the other hand, an embodiment of the present invention provides a DNA fragment comprising: a promoter, loxP-Stop-loxP, SARS-CoV-2N CDS, WPRE, and a poly-A gene; the nucleotide sequence of the SARS-CoV-2N CDS is shown in SEQ ID NO: 3:

[0472]

[0473] The present invention adds a post-transcriptional regulatory sequence WPRE from woodchuck hepatitis virus before polyA, which can improve the stability of over-expressed gene RNA, thereby increasing the expression level of the N gene.

[0474] In some embodiments, the nucleotide sequence of the DNA fragment is shown in SEQ ID NO: 4:

[0475]

[0476] In another aspect, an embodiment of the present invention provides a vector comprising the DNA fragment described in any of the aforementioned embodiments.

[0477] In some embodiments, the vector can be used as a targeting vector. This application utilizes sequence-independent ligation cloning (SLIC) technology to construct a homologous recombination vector carrying a conditional overexpression N sequence, and the recombination system used in the construction process is a Cre-LoxP inducible expression system.

[0478] In some embodiments, the vector further comprises: a 5' homology arm (5' homology arm, 5'arm) and a 3' homology arm (3' homology arm, 3'arm) of the safety site, which are used to directionally clone the DNA fragment into the safety site, wherein the 5' homology arm is located at the 5' end of the DNA fragment, and the 3' homology arm is located at the 3' end of the DNA fragment. The homology arm sequences at both ends of the vector serve as templates for homologous recombination repair and are co-injected into mouse fertilized eggs with Cas9 / sgRNA, increasing the probability of homologous recombination repair and the probability of obtaining positive animals (such as mice). The prepared animal model does not contain other exogenous sequences other than the target sequence, avoiding the risk of random insertions or deletions during repair.

[0479] In some embodiments, the safe site is the Tigre site. The Tigre site on mouse chromosome 9 is selected as the specific insertion site for the target gene. Because this site belongs to a safe region, it avoids interference with endogenous gene expression, and the site-specific insertion of the exogenous gene will not affect the expression of other genes.

[0480] In some embodiments, the nucleotide sequence of the targeting vector is shown in SEQ ID NO:5.

[0481]

[0482] On the other hand, an embodiment of the present invention provides a method for constructing an animal model, which comprises: injecting Cas9 protein or Cas9 mRNA, sgRNA designed for the Tigre site, and the vector described in any of the preceding embodiments into the cytoplasm or nucleus of a fertilized egg, transplanting it into a surrogate animal mother, and screening the target animals as the F0 generation.

[0483] The animal model provided by the present invention has a complete immune system and is suitable for all studies on the interaction between SARS-CoV-2N protein and the immune system.

[0484] The present invention achieves single-copy target gene insertion, making it easier to predict expression levels.

[0485] The construction method further comprises: mating the F0 generation animals with background animals to obtain the F1 generation.

[0486] In some embodiments, the sgRNA includes any one or more combinations of the sequence shown in SEQ ID No: 6 (CATCCCAAAGTTAGGTGTTA), the sequence shown in SEQ ID No: 7 (ACAGAAAACATCCCAAAGTT), and the sequence shown in SEQ ID No: 8 (Actgccataacacctaactt).

[0487] In some embodiments, the animal is a mouse.

[0488] In some embodiments, the background animal is a mouse expressing hACE2 (the receptor for SARS-CoV-2). A dual-target model expressing the N protein is constructed based on a humanized hACE2 (the receptor for SARS-CoV-2) background mouse. This dual-target model is capable of being infected by SARS-CoV-2 and can be used for research on the development of preventive and therapeutic drugs for COVID-19.

[0489] In some embodiments, the background animal is an H11-K18-hACE2 mouse.

[0490] In some embodiments, the fertilized egg is a fertilized egg of a K18-hACE2 KI mouse.

[0491] In some embodiments, the screening of target animals includes: screening animals that are double positive for SARS-Cov-2 N and hACE2.

[0492] In some embodiments, the construction method further includes: mating the F1 generation positive mice or their positive offspring with transgenic animals expressing CRE throughout the body or in specific tissues, or infecting the F1 generation positive mice or their positive offspring with Cre virus to obtain an animal model expressing SARS-Cov-2 N and hACE2.

[0493] The animal model provided by the present invention can specifically and controllably express the SARS-CoV-2N protein, solving the problem of expressing the SARS-CoV-2N protein in specific tissues or at specific times. The "promoter-loxP-Stop-loxP-Kozak-SARS-CoV-2N-WPRE-polyA" sequence is inserted into the Tirge site. Since the promoter and the target gene N are separated by a loxP-Stop-loxP structure, under normal circumstances, although the mouse contains the N gene, the promoter cannot initiate the expression of the N gene. After mating with tissue-specific Cre tool mice, the offspring will express the N protein in specific tissues, or by using Cre virus infection, the mice can be controlled to express the N protein at a specific time.

[0494] This animal model can controllably express N protein in the absence of infection, and can be used to simulate the role of N protein in inducing inflammatory response, cytokine storm, and immune escape during SARS-CoV-2 infection. It is of great significance for studying the development mechanism of COVID-19 and can promote the research of new coronavirus drugs targeting N protein.

[0495] At the same time, using the novel coronavirus SARS-CoV-2 trans-complementation replicon system lacking the N gene (the replicon system described in any of the aforementioned embodiments), a mouse model that infects tissue-specifically or expresses the SARS-CoV-2N protein at a specific time was successfully constructed to develop a safe novel coronavirus SARS-CoV-2 animal infection model that can be used in a BSL-2 laboratory.

[0496] In some embodiments, the construction method further comprises: using the viral replicon described in any of the preceding embodiments to infect the animal model expressing SARS-Cov-2 N and hACE2 or its progeny to obtain a systemic or tissue-specific animal infection model, which can be used for the evaluation of neutralizing antibodies and / or antiviral drugs.

[0497] In addition, an embodiment of the present invention provides the use of an animal model constructed by the method for constructing an animal model as described in any of the preceding embodiments in screening drugs against the new coronavirus SARS-Cov-2.

[0498] The features and performance of the present invention are further described in detail below with reference to the embodiments.

[0499] Example 1: Construction method of novel coronavirus replicon

[0500] 1.1 The genome sequence of the novel coronavirus SARS-CoV-2 BA.5.2 mutant strain was divided into 7 segments (A, B, C, D, E, F, G), in which the SARS-CoV-2 N gene sequence was replaced by the GFP sequence. These 7 segments were synthesized and cloned into the pUC57 vector and named pUC57-A, pUC57-B, pUC57-C, pUC57-D, pUC57-E, pUC57-F, pUC57-G ( Figure 1 The pUC57-A plasmid contains a bacteriophage T7 promoter sequence at the 5' end of the SARS-CoV-2 5'-UTR, while the pUC57-F plasmid contains the SARS-CoV-2 S gene sequence. CEN / ARS-TRP was also synthesized and cloned into the pUC57 vector. Sequence information is shown in Table 1.

[0501] Table 1 Sequence

[0502]

[0503]

[0504]

[0505]

[0506]

[0507] 1.2 Primer design and fragment amplification

[0508] A pair of primers were designed at the head and tail of sequences A, B, C, D, E, F, and G to amplify the fragments. The resulting PCR products had 60 bp overlapping sequences between adjacent fragments.

[0509] pCC1-Af:gtaaaacgacggccagtgaattgtaatacgactcactatagccaggtaacaaaccaacc;

[0510] ABf:gctgctaacttttgtgcacttatcttagcctactgtaataagacagtaggtgagttaggt;

[0511] ABr:acctaactcacctactgtcttattacagtaggctaagataagtgcacaaaagttagcag;

[0512] BCf:accaatgtgctatgagacacaatttcactattaagggttcattccttaaatggttcatgt;

[0513] BCr:acatgaaccattaaggaatgaacccttaatagtgaaattgtgtctcatagcacattggt;

[0514] CDf:actggataccacttcagagagctaggtgttgtacataatcaggatgtaaacttacatag;

[0515] CDr:ctatgtaagtttacatcctgattatgtacaacacctagctctctgaagtggtatccagt;

[0516] DEf:actaccaactcaaactgttgattcatcacagggctcagaatatgactatgtcatattca;

[0517] DEr:tgaatatgacatagtcatattctgagccctgtgatgaatcaacagtttgagttggtagt;

[0518] EFf:atgattttatctcttcttagtaaaggtagacttataattagagaaaacaacagagttgt;

[0519] EFr:acaactctgttgttttctctaattataagtctacctttactaagaagagataaaaatcat;

[0520] FGf:gaacttatggatttgtttatgagaatcttcacaattggaactgtaactttgaagcaagg;

[0521] FGr:ccttgcttcaaagttacagttccaattgtgaagattctcataaacaaatccataagttc;

[0522] G-CEN / ARS-TRP-f:aggagaatgacaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaagcggccgcgatcgcttgcctgtaacttacac;

[0523] G-CEN / ARS-TRP-r:gtgtaagttacaggcaagcgatcgcggccgcttttttttttttttttttttttttttttttgtcattctcct;

[0524] CEN / ARS-Trp-pCC1-r:ctagaggatccccgggtaccgagctaacgacattactatatatataatataggaagcatttaatag.

[0525] It will be understood that the direction of the nucleotide sequences described herein is 5' to 3'.

[0526] Fragment A was amplified by PCR using the pUC57-A vector as a template using the primer pair pCC1-Af and ABr; fragment B was amplified by PCR using the pUC57-B vector as a template using the primer pair ABf and BCr; fragment C was amplified by PCR using the pUC57-C vector as a template using the primer pair BCf and CDr; fragment D was amplified by PCR using the pUC57-D vector as a template using the primer pair CDf and DEr; fragment D was amplified by PCR using the pUC57-E vector as a template using the primer pair DEf and EFr. Fragment E was obtained by PCR amplification using primers EFf and FGr with pUC57-F as template; Fragment G was obtained by PCR amplification using primers FGf and G-CEN / ARS-TRP-r with pUC57-G as template; CEN / ARE-Trp fragment with homology arms was obtained by PCR amplification using primers G-CEN / ARS-TRP-f and CEN / ARS-Trp-pCC1-r with pUC57-CEN / ARE-Trp as template. Figure 2 ).

[0527] 1.3. Using the lithium acetate method to transform yeast, the pCC1-BAC vector linearized by BamHI and the 8 PCR fragments obtained in the above steps were transformed into yeast MaV203 competent cells, and the positive clones were screened by colony PCR to obtain the pCC1-Trp-SARS-CoV-2 vector ( Figure 3 ).

[0528] 1.4. Linearize pCC1-Trp-SARS-CoV-2 with NotI and use it as a template for in vitro transcription to obtain full-length genomic mRNA. Design a pair of primers, T7-NF and N-polyAR, and perform PCR using the pCDNA3.1-SARS-CoV-2-N plasmid as a template. Use the purified PCR product as a template to in vitro transcribe SARS-CoV-2-N mRNA. 1 μg of full-length mRNA and SARS-CoV-2-N mRNA were each electrophoresed to test RNA integrity ( Figure 4 ). The primer numbers and sequences are as follows:

[0529] T7-N F:ccagtaatacgactcactatagatgtctgataatggaccccaaaatca;

[0530] N-polyA R:tttttttttttttttttttttttttaggcctgagttgagtcagcact.

[0531] 1.5. Construction of a Vero E6 cell line stably expressing the novel coronavirus SARS-CoV-2 N protein: The plasmids pLVX-SARS-CoV-2-N-puro, pMD2.G, and psPAX2 were transfected into HEK293T cells. After 48 hours, the cell supernatant was collected and filtered through a 0.45 μm filter. 1 mL of the filtered supernatant was used to infect Vero E6 cells plated one day in advance in a 6-well plate at a density of approximately 60%. Polybrene was also added at a final concentration of 10 μg / ml. After 24 hours of culture, the culture medium was replaced with fresh medium containing 20 μg / ml puromycin for cell selection, ultimately obtaining Vero E6-N cells stably expressing the novel coronavirus SARS-CoV-2 N protein.

[0532] 1.6. Electroporate the mRNA obtained in step 1.4 above into 1x10 7 After 16 hours, GFP signal can be observed in Vero E6-N cells ( Figure 5 ) and collect the supernatant of the electroporated cells after 48 hours. The supernatant containing the replicon collected at this time is recorded as P0. After centrifugation, 1 mL of P0 supernatant is added to six-well plates of Vero E6-N cells with a cell confluence of 60%-80%. GFP signal can be seen in 12-24 hours ( Figure 6 ), and after 48-72 hours, the supernatant containing the replicon was collected and recorded as P1. It was aliquoted and stored at -80°C. The supernatant obtained in this step can be used in subsequent applications.

[0533] 2. Screening of replicon reporter genes and their combinations:

[0534] 2.1. Design primers to replace the GFP sequence in the pUC57-G plasmid with different dual reporter genes. The primer numbers and sequences are as follows:

[0535] pUC57-G F:gcgaatgcatctagatatcgacaattggaactgtaactttgaagca;

[0536] Fluc-R1:aatgttcttagcatcggccattttagtttgttcgtttagatgaaatc;

[0537] Rluc-R1:atcatacactttgctggtcattttagtttgttcgtttagatgaaatc;

[0538] Nanoluc-R1:tcttcgagtgtgaagaccattttagtttgttcgtttagatgaaat;

[0539] Fluc-F1:gatttcatctaaacgaacaaactaaaatggccgatgctaagaacatt;

[0540] Rluc-F1:gatttcatctaaacgaacaaactaaaatgaccagcaaagtgtatgat;

[0541] Nanoluc-F1:gatttcatctaaacgaacaaactaaaatggtcttcacactcgaaga;

[0542] Fluc-GFP R2:tcgggcttgatcacgcccatgccgctagagcctaatgttcttagcatcggccat;

[0543] Rluc-GFP R2:tcgggcttgatcacgcccatgccgctagagccgccctgttcgtttttcagcac;

[0544] Nanoluc-GFP R2:tcgggcttgatcacgcccatgccgctagagcccgccagaatgcgttcgc;

[0545] Fluc-GFP F2:atggccgatgctaagaacaggctctagcggcatgggcgtgatcaagcccga;

[0546] Rluc-GFP F2:gtgctgaaaaacgaacagggcggctctagcggcatgggcgtgatcaagcccga;

[0547] Nanoluc-GFP F2:gcgaacgcattctggcgggctctagcggcatgggcgtgatcaagcccga;

[0548] GFP-F3:taccccgccaggccggctaaactcatgcagaccacacaag;

[0549] GFP-R3:cttgtgtggtctgcatgagtttagccggcctggcggggta;

[0550] pUC57-G R:ctatgaccatgattacgccatgtcattctcctaagaagctattaa;

[0551] The reporter genes Flu, Rluc, and Nanoluc were isolated from plasmids by PCR, and the HibiT sequence was inserted using primers. The pUC57-G-Fluc-GFP, pUC57-G-Rluc-GFP, pUC57-G-Nanoluc-GFP, and pUC57-G-GFP-HibiT plasmids were constructed using the Multi-Fragment One-Step Rapid Cloning Kit (Yisheng Biotechnology). The sequences of the G-Fluc-GFP, G-Rluc-GFP, G-Nanoluc-GFP, and pUC57-G-GFP-HibiT fragments are shown in SEQ ID Nos. 17 to 20.

[0552]

[0553]

[0554]

[0555] 2.2 Using the plasmids obtained in the above steps as templates, follow steps 1.2 to 1.6 to finally obtain four new coronavirus SARS-CoV-2-Flu-GFP dual reporter gene replicons: SARS-CoV-2-Flu-GFP, SARS-CoV-2-Rlu-GFP, SARS-CoV-2-Nanoluc-GFP, and SARS-CoV-2-GFP-HibiT.

[0556] 2.3 Testing reporter gene stability and signal strength: The four dual reporter gene replicons obtained in the above steps were serially passaged for five generations in Vero E6-N cells at a multiplicity of infection of 0.05 (MOI = 0.05), centrifuged at 500 rpm for 5 minutes, and the supernatants were collected and recorded as P1, P2, P3, P4, and P5, respectively.

[0557] Vero E6-N cells were cultured at 1x10 4 Cells were seeded into a white 96-well plate at a density of 100 cells / well. After 16-24 hours, the cell confluence reached 80%. VeroE6-N cells were infected with four different dual reporter gene replicons from generations P1 to P5 at an MOI of 0.05 (three parallel wells were set up for each group). After incubation at 37°C for 2 hours, the virus-containing medium in each well was aspirated and each well was washed 2-3 times with PBS. 100 μl of 5% FBSDMEM medium was added to each well and cultured for 48 hours at 37°C and 5% CO2. After 48 hours, the corresponding cell lysate and fluorescent substrate were added to the replicon-infected 96-well plates according to the Promega instructions. The corresponding luminescence values ​​were read on a multi-function microplate reader and graphed using Prism Graphpad (see Figure 7 ).

[0558] Take 100 μl of the supernatant of P1 and P5 generations of four replicons respectively, use Trizol LS to extract the supernatant RNA of the replicon, design primers RT-F and RT-R (the two ends of N are OK) to perform RT-PCR, and detect the length of the P1 and P5 generation reporter gene PCR products ( Figure 8 ).

[0559] The above results show that among the four dual reporter genes screened, the Nanoluc-GFP dual reporter gene has the best luminescence intensity and stability in the new coronavirus replicon.

[0560] 3. Preparation of high titer replicons:

[0561] Plasmids pLVX-TMPRSS2-BSD, pMD2.G, and psPAX2 were transfected into HEK293T cells. After 48 hours, the cell supernatant was collected and filtered through a 0.45μm filter. 1 mL of the filtered supernatant was used to infect Vero-E6-N cells plated one day in advance in a 6-well plate at a density of approximately 60%. Polybrene was also added at a final concentration of 10μg / ml. After 24 hours of culture, fresh medium containing 20μg / ml blasticidin was replaced for cell selection, ultimately obtaining Vero E6-N-TMPRSS2 cells that stably express the novel coronavirus SARS-CoV-2 N protein and transmembrane serine protease.

[0562] Vero E6-N cells and Vero E6-N-TMPRSS2 cells were cultured at 3×10 5 Cells were seeded into 6-well plates at a density of 100 cells / well, reaching 80% cell confluency after 16-24 hours. The cells were infected with the novel coronavirus SARS-CoV-2-Nanoluc-GFP replicon at an MOI of 0.05 according to the groupings in Table 2. After incubation at 37°C for 2 hours, the virus-containing medium was aspirated from each well and each well was washed 2-3 times with PBS. Each well was supplemented with fresh 5% FBSDMEM medium containing various concentrations of trypsin (TPCK-treated trypsin) and incubated for an additional 48 hours at 37°C in 5% CO2.

[0563] Table 2 Cell infection

[0564]

[0565] The supernatant was collected and the TCID of the novel coronavirus SARS-CoV-2-Nanoluc-GFP replicon in the supernatant was determined and calculated according to the Reed-Muench method. 50 The results are as follows Figure 9 The results show that when the culture medium contains 20 μg / ml trypsin, the highest titer of 1.2x10 7 TCID 50 / ml of the novel coronavirus SARS-CoV-2-Nanoluc-GFP replicon.

[0566] Example 2: Construction of a mouse infection model of the novel coronavirus SARS-CoV-2 replicon

[0567] 1. Construction and breeding of hACE2 and SARS-CoV-2-N transgenic mice

[0568] 1.1 Construction and comparison of SARS-CoV-2-N transgenic mice

[0569] like Figure 10 As shown, three types of hACE2 and SARS-CoV-2-N transgenic mice were constructed.

[0570] Model Animal A: In the context of B6 / JGpt-H11em1Cin(K18-ACE2) / Gpt(K18-hACE2 KI) transgenic mice (Jicui Yaokang), the CAG-LSL-SARS-CoV-2-N-Wpre-PolyA gene fragment was site-specifically inserted into the Tigre locus of the mouse using CRISPR / Cas9 technology. That is, the vector was constructed in vitro, and the CRISPR / Cas9 and Donor vector were microinjected into the fertilized eggs of K18-hACE2 KI mice to obtain F0 generation mice. The correct F0 generation positive mice verified by PCR and sequencing were mated with H11-K18-hACE2 mice to obtain a stable and heritable F1 generation positive mouse model (see step 1.2 of this example for details).

[0571] Model Animal B: The CAG-LSL-SARS-CoV-2-N-P2A-hACE2-Wpre-PolyA gene fragment was site-specifically inserted into the H11 locus of mice using CRISPR / Cas9 technology. This construct was constructed in vitro, and the CRISPR / Cas9 and donor vector were microinjected into fertilized eggs of C57BL / 6JGpt mice to generate F0 generation mice. F0 generation positive mice, verified by PCR and sequencing, were mated with wild-type mice to generate a stable, heritable F1 generation positive mouse model.

[0572] Model Animal C: The CAG-LSL-SARS-CoV-2-N-Wpre-PolyA-anti-[CAG-hACE2-PolyA] gene fragment was site-specifically inserted into the H11 locus of mice using CRISPR / Cas9 technology. The vector was constructed in vitro, and the CRISPR / Cas9 and donor vector were microinjected into fertilized eggs of C57BL / 6JGpt mice to generate F0 generation mice. F0 generation positive mice, verified by PCR and sequencing, were mated with wild-type mice to generate a stable F1 generation positive mouse model.

[0573] SA-CreERT2 transgenic tool mice were hybridized with the above three model animals respectively. After PCR verification, the corresponding SA-CreERT2-N-hACE2 transgenic mice were obtained, which were named SA-CreERT2N-hACE2-A, SA-CreERT2-N-hACE2-B, and SA-CreERT2-N-hACE2-C respectively. Tamoxifen was dissolved in corn oil and injected intraperitoneally into SA-CreERT2-N-hACE2 mice of appropriate age at a dose of 75 mg / kg to induce Cre expression. The injection was given once every 24 hours. After five consecutive days of injection, the mice were kept for 7 days. The mouse lungs were then taken and the expression levels of hACE2 and SARS-CoV-2-N in the mouse lungs were detected by fluorescent quantitative PCR ( Figure 11 ). Fluorescence quantitative PCR results showed that among the three SA-CreERT2-N-hACE2 mice induced by tamoxifen, the expression levels of hACE2 and SARS-CoV-2-N in SA-CreERT2-N-hACE2-A mice were the highest. Therefore, SARS-CoV-2-N transgenic mice constructed in the K18-hACE2 KI background (model animal A) were preferred.

[0574] 1.2 Construction of homologous DNA donor for conditional overexpression of SARS-CoV-2N gene targeting in model animal A

[0575] SLIC ligation was used to construct a homologous recombination vector carrying a conditional overexpression N sequence. The recombination system used was the Cre-LoxP inducible expression system. The vector included: safety site 5' homology arm sequence-pCAG-loxP-Stop-loxP-Kozak-SARS-CoV-2N-WPRE-polyA-3' homology arm sequence. The donor element structure was as follows: Figure 12 Among them, the coding region gene fragment of the SARS-CoV-2N gene was queried from NCBI and synthesized, as shown in SEQ ID NO: 3.

[0576] 1.3Tirge site sgRNA design

[0577] Determine the approximate sgRNA region based on the Tirge safe sites. Using sgRNA evaluation software, select the sequences with the lowest off-target rates for the target fragment and identify the 5' and 3' target sites. The sequences listed in the table below are used as sgRNAs. Synthesize and construct the sgRNA expression vector puc57-sgRNA.

[0578] Table 3 gRNA sequences

[0579]

[0580] sgRNA transcript preparation method: PCR was performed using the PrimerStar or PrimerStar Max system, sgRNA-F and sgRNA-R as primers, and the correctly sequenced puc57-sgRNA plasmid (1:30 dilution) as a template. The PCR product was purified to prepare the sgRNA transcription template. sgRNA was transcribed using the T7-ShortScript in vitro transcription kit (AM1354).

[0581] In this example, transcription of three sgRNAs was performed simultaneously.

[0582] 1.4 Establishment of a mouse model with conditional overexpression of the SARS-CoV-2N gene

[0583] The sgRNA designed above was mixed with the conditionally overexpressed SARS-CoV-2N targeting homologous DNA donor and Cas9 protein, injected into 0.5-day-old background mouse fertilized eggs, and transplanted into 0.5-day-old pseudo-pregnant female mice. After the mice were born, the targeted mice were screened by genetic identification.

[0584] Genotype identification of the F0 generation of mice: The tail genomic DNA of the obtained F0 mice was subjected to two pairs of primers for end-to-end PCR identification after targeting. The primers 0X0447-01-Tigre-5tF1 / H11-CAG-5tR2 were located outside the 5' homology arm and inside the insert of the targeting vector, respectively. If this primer pair amplifies and produces a PCR product, it indicates that the target vector has been effectively inserted into the 5' region of the mouse genome. The primers BGH-pA-tF1 / 0X0447-01-Tigre-3tR11 are located inside the insert of the targeting vector and outside the 3' homology arm, respectively. If this primer pair amplifies and produces a PCR product, it indicates that the target vector has been effectively inserted into the 3' region of the mouse genome.

[0585] Table 4. F0 identification of SARS-CoV-2N correct insertion primers

[0586]

[0587]

[0588] Optional simplified protocol for F1 mouse offspring:

[0589] Table 5. F1 primers for identifying the correct insertion of SARS-CoV-2N

[0590]

[0591] Table 6. Primers for identifying hACE2 background mice

[0592]

[0593] Note: KI is the on-target genotype; WT is the wild type; KI / KI is the homozygous; KI / WT is the heterozygous.

[0594] The PCR reaction system and reaction conditions are shown in the following table:

[0595] Table 7. PCR reaction system

[0596] Reagent (Vazyme P112-03) Volume (μl) Specification 2×Taq Master Mix, Dye Plus 12.5 \ ddH2O 9.5 \ Primer A (10 pmol / μl) 1 10 μM Primer B (10 pmol / μl) 1 10 μM Template (≈100 ng / μl) 1 \

[0597] Table 8. PCR reaction conditions

[0598]

[0599] Mouse clones that were positive by PCR at both ends were sequenced for verification. The sequencing primer information is shown in the table below. After the targeting vector was correctly inserted into the mouse genome, the clones that were correctly sequenced were identified as positive F0 mice.

[0600] Table 9. Sequencing primers

[0601]

[0602] F0 identification results:

[0603] Based on the H11-K18-hACE2 mouse (C57BL / 6JGpt) background, one positive F0 mouse was obtained. The results of SARS-CoV-2N-KI-target mouse tail DNA identification are as follows Figure 13 As shown, the results of tandem detection are as follows Figure 14 As shown, the background hACE2-KI-target mouse tail DNA identification results are as follows Figure 15 As shown (WT control is C57BL / 6JGpt genomic DNA; N is a blank control, a control without template; P is a positive control; M is a TRANS2K PLUS II band: 8000bp\5000bp\3000bp\2000bp\1000bp\750bp\500bp\250bp\100bp). The 5' and 3' ends of the SARS-CoV-2 N and hACE2 genes in mice #2 and #3 were positive for identification and sequencing, and no tandem replication was observed in #2. Mouse #2 was selected as the F0 positive mouse that underwent correct genetic recombination.

[0604] F1 identification results:

[0605] SARS-CoV-2N and hACE2 double-positive F0 mice were bred with H11-K18-hACE2-C57BL / 6JGpt background mice to obtain F1 generation. The SARS-CoV-2N and hACE2 genes were identified in the tails of F1 generation mice. The PCR test results of F1 generation mice are shown in the table. Figure 16 (From top to bottom, electropherograms of the 5' and 3' ends of the SARS-CoV-2N-KI-target, tandem detection, and background hACE2-KI-target 5' and 3' ends for identification. WT uses C57BL / 6JGpt genomic DNA as a template; N is a blank control, a no-template control; P is a positive control; TRANS2K PLUS II bands: 8000bp, 5000bp, 3000bp, 2000bp, 1000bp, 750bp, 500bp, 250bp, and 100bp.) Mouse #42 showed positive results for both the 5' and 3' ends of the SARS-CoV-2N gene and the 5' and 3' ends of the background hACE2, with no tandem detection, indicating that mouse #42 was a positive mouse with correct genetic recombination. After extensive F1 breeding, interbreeding was performed to obtain mice homozygous for both SARS-CoV-2N and hACE2.

[0606] 1.5 Construction of systemic and tissue-specific mouse infection models

[0607] The lung-specific expression of Sftpc-IRES-iCre and the systemic expression of SA-CreERT2 transgenic tool mice were mated with the F1 generation positive mice of the above K18-hACE2 KI / Tigre-N, and the positive mice verified by PCR were self-fertilized for one generation. Finally, homozygous transgenic mice were obtained by PCR verification and named Sftpc-hACE2-N. F / F and SA-CreERT2-hACE2-N F / F Tamoxifen was dissolved in corn oil and injected intraperitoneally into SA-CreERT2-hACE2-N mice of appropriate age at a dose of 75 mg / kg. F / F Inducible Cre expression in mice ( Figure 17 ), injected once every 24 hours for five consecutive days and then the mice were kept for 7 days. F / F Cre expression is achieved without the need for tamoxifen induction.

[0608] 1x10 5 TCID5 of SARS-CoV-2-Nanoluc-GFP replicon was used to infect the above-mentioned SA-CreERT2-hACE2-N cells induced by tamoxifen by intranasal drip. F / F Mouse and Sftpc-hACE2-N F / F At the same time, a control group was set up, and an equal volume of PBS was dripped into the nose of the Sftpc-hACE2-N cells induced by tamoxifen. F / F and SA-CreERT2-hACE2-N F / F Mice. The mice were weighed daily for 7 days ( Figure 18 On the 7th day, the mice were euthanized, and the lungs and other organs were removed. After grinding and homogenization, the levels of SARS-CoV-2-E RNA in each organ were detected by fluorescence quantitative PCR ( Figure 19 ), while immunohistochemistry and analysis of lung and brain tissues were performed ( Figure 20 ).

[0609] The experimental results showed that the constructed transgenic mice can efficiently express hACE2 and the new coronavirus SARS-CoV-2N protein, and support the replication of the new coronavirus replicon SARS-CoV-2-Nanoluc-GFP.

[0610] Example 3: Application of the novel coronavirus SARS-CoV-2 replicon animal infection model in the evaluation of neutralizing antibodies and antiviral drugs

[0611] Tamoxifen was dissolved in corn oil and injected intraperitoneally into SA-CreERT2-hACE2-N mice of appropriate age at a dose of 75 mg / kg. F / F The mice were induced to express Cre and injected once every 24 hours for five consecutive days and then kept for another 7 days.

[0612] The induced mice were divided into four groups and treated according to the following methods.

[0613] REGEN-COV2 treatment group: 1x10 5 Four hours after intranasal infection with TCID5 SARS-CoV-2-Nanoluc-GFP replicon, 10 mg / Kg neutralizing antibody REGN-COV2 was injected intraperitoneally.

[0614] Paxlovid-treated mice were orally administered 5 mg / kg of Paxlovid two hours prior to the tamoxifen-induced treatment. Mice were then intranasally infected with 10,000 PFU of SARS-CoV-2-Nanoluc-GFP replicons. The same dose of Paxlovid was administered orally every 12 hours for seven days.

[0615] Infected group: 1x10 5 Mice were infected intranasally with TCID5 SARS-CoV-2-Nanoluc-GFP replicon.

[0616] Blank (mock) group: no infection and no other treatment.

[0617] Four groups of mice were weighed daily for seven days ( Figure 21On the seventh day, the mice were euthanized, and the lungs were removed and homogenized. The levels of SARS-CoV-2-E RNA in the lung and brain tissues were detected by quantitative fluorescence PCR ( Figure 22 ), and H&E staining analysis was performed on lung and brain tissues ( Figure 23 ).

[0618] These results suggest that the REGN-COV2 antibody and the small molecule drug remdesivir can reduce viral load in infected mice and alleviate damage to their lung and brain tissue. This animal infection model of the novel coronavirus replicon can be used to evaluate neutralizing antibodies and antiviral drugs.

[0619] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A viral replicon, characterized in that It includes: A novel coronavirus SARS-Cov-2 genome sequence in which the N gene of the novel coronavirus SARS-Cov-2 is replaced by a reporter gene, wherein the reporter gene includes a Nanoluc reporter gene and a GFP dual reporter gene; Optionally, the replicon further comprises a promoter, and the promoter is located upstream of the 5'UTR of the novel coronavirus SARS-Cov-2 genome; Optionally, the promoter comprises bacteriophage promoter T7; Optionally, the nucleotide sequence of the replicon is shown as SEQ ID No:

1.

2. A DNA molecule, characterized in that It includes: A nucleotide sequence capable of rescuing the viral replicon of claim 1; Optionally, the DNA molecule comprises the nucleotide sequence shown in SEQ ID NO:

2.

3. A viral replication subsystem, characterized in that: These include: Stability A cell expressing the novel coronavirus SARS-CoV-2N protein and an expression vector containing the viral replicon according to claim 1 or the DNA molecule according to claim 2.

4. The viral replicon system according to claim 3, characterized in that The expression vector is located in the cell; Optionally, the cell is a cell that stably expresses the new coronavirus SARS-CoV-2N protein and transmembrane serine protease.

5. A method for producing a viral replicon, characterized in that: It includes: training The cell stably expressing the novel coronavirus SARS-CoV-2N protein as described in claim 3 or 4; Optionally, the culturing comprises: adding trypsin to the culture environment of the cells; Optionally, the trypsin has an action concentration of 1 to 40 μg / ml in the culture environment; Optionally, the trypsin has an action concentration of 15-25 μg / ml in the culture environment, preferably 20 μg / ml.

6. Use of the viral replicon according to claim 1, the DNA molecule according to claim 2, the viral replicon system according to claim 3 or 4, or the viral replicon produced by the production method according to claim 5 in drug screening against the new coronavirus SARS-Cov-2.

7. A DNA fragment, characterized in that It includes: promoter, loxP-Stop-loxP, SARS-CoV-2N CDS, WPRE, and poly-A genes; The nucleotide sequence of the SARS-CoV-2N CDS is shown in SEQ ID NO: 3; Optionally, the nucleotide sequence of the DNA fragment is shown in SEQ ID NO:

4.

8. A carrier, characterized in that It contains the DNA fragment according to claim 7; Optionally, the vector further comprises: a 5' homology arm and a 3' homology arm of the safety site, wherein the 5' homology arm is located at the 5' end of the DNA fragment, and the 3' homology arm is located at the 3' end of the DNA fragment; Optionally, the safety site is a Tigre site; Optionally, the nucleotide sequence of the vector is as shown in SEQ ID NO:

5.

9. A method for constructing an animal model, characterized in that: It includes: Injecting Cas9 protein or Cas9 mRNA, sgRNA designed for the Tigre site, and the vector described in claim 8 into the cytoplasm or nucleus of a fertilized egg, transplanting it into a surrogate mother, and screening the targeted animals as the F0 generation; The construction method further comprises: mating the F0 generation animal with the background animal to obtain the F1 generation; Optionally, the sgRNA includes any one or more combinations of the sequence shown in SEQ ID No: 6, the sequence shown in SEQ ID No: 7, and the sequence shown in SEQ ID No: 8; Optionally, the animal is a mouse; Optionally, the background animal is a mouse expressing hACE2; Optionally, the background animal is an H11-K18-hACE2 mouse; Optionally, the fertilized egg is a fertilized egg of a K18-hACE2 KI mouse; Optionally, the screening of target animals comprises: screening animals that are double positive for SARS-Cov-2 N and hACE2; Optionally, the construction method further comprises: mating the F1 generation positive mice or their positive offspring with transgenic animals expressing CRE throughout the body or in specific tissues, or infecting the F1 generation positive mice or their positive offspring with Cre virus to obtain an animal model expressing SARS-Cov-2 N and hACE2; Optionally, the construction method further comprises: using the viral replicon described in claim 1 to infect the animal model expressing SARS-Cov-2 N and hACE2 or its progeny to obtain a systemic or tissue-specific animal infection model.

10. Use of the animal model obtained by the method for constructing an animal model as described in claim 9 in screening drugs against the new coronavirus SARS-Cov-2.

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